CN114561283A - Full-automatic nucleic acid extraction, constant temperature amplification detect all-in-one - Google Patents
Full-automatic nucleic acid extraction, constant temperature amplification detect all-in-one Download PDFInfo
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Abstract
The invention discloses a full-automatic nucleic acid extraction and constant temperature amplification detection integrated machine, which comprises: the nucleic acid extraction and amplification integrated device comprises a nucleic acid extraction and amplification container, a centrifuge and a heating module; and an optical detection module; the nucleic acid extraction and amplification container comprises a bracket and two centrifugal container tubes, wherein the centrifugal container tubes comprise outer sleeves, centrifugal tubes, cracking tubes, a plurality of pre-sealing liquid bags, centrifugal columns and collecting tubes; the bottom of the pre-sealing liquid bag is provided with a liquid outlet, and the liquid outlet is sealed by a paraffin valve. The invention can realize automatic nucleic acid extraction, purification, isothermal amplification and detection, and avoids the defects of complex control, large equipment volume, incapability of effectively shortening the time and the like caused by the adoption of a manipulator for automatic reagent addition operation.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a full-automatic nucleic acid extraction and constant-temperature amplification detection all-in-one machine.
Background
Nucleic acid amplification detection technology is widely applied to various fields of life sciences such as molecular biology, medicine, law and the like, and because PCR requires continuous temperature change to realize nucleic acid amplification, the limitation of depending on excellent instruments and equipment cannot be got rid of all the time, and nucleic acid isothermal amplification technology with various mechanisms is generated and is also developed rapidly. Among them, Loop-mediated isothermal amplification (LAMP) has been developed since 2000, and has been widely used in recent years for detecting microorganisms such as viruses and bacteria due to its characteristics of rapidness (within 1 hour), simplicity (only 65 ℃ temperature control equipment), sensitivity, specificity, etc., and thus makes a great contribution to the rapid detection of microorganisms on a large scale. For example, patent CN111270010A discloses a loop-mediated isothermal amplification detection primer set for detecting 2019-nCoV and application thereof.
The detection method of the nucleic acid isothermal amplification mainly comprises the following steps: nucleic acid extraction and purification, isothermal amplification and optical detection, which have many steps, especially nucleic acid extraction and purification steps, and large workload of operators, make some automated nucleic acid isothermal amplification detection equipment have great demands. A centrifugal column method for extracting nucleic acid is one of the mainstream nucleic acid extraction methods at present, and the basic principle is that a lysate is utilized to promote cell disruption, so that nucleic acid in cells is released, then the released nucleic acid is specifically adsorbed on a nucleic acid adsorption membrane (such as a silica gel membrane) of a centrifugal column, the membrane only has strong affinity and adsorption force on the nucleic acid, and basically does not adsorb other biochemical components such as protein, polysaccharide and lipid, so that the nucleic acid can be thrown out of the column during centrifugation, the centrifugal column is rinsed by rinsing liquid to remove impurities, and finally the nucleic acid adsorbed on the nucleic acid adsorption membrane is eluted by the rinsing liquid, so that purified nucleic acid can be obtained for subsequent nucleic acid amplification detection.
In some automatic nucleic acid detection apparatuses disclosed at present, a manipulator is usually used to add reagents at various stages in a detection process, such as lysis solution, binding solution, rinsing solution, eluent, amplification system solution, and the like. For example, patent CN208454938U discloses a nucleic acid extraction module for a full-automatic nucleic acid extraction amplification and diagnosis integrated machine. Such automated nucleic acid extraction amplification methods still suffer from the following drawbacks: 1. the operation process is still very complicated, the control of the mechanical arm is relatively complicated, and more importantly, when the mechanical arm adds the reagent, the centrifugal operation needs to be stopped, and the reagent can be centrifuged again after the reagent is added, so that the time of the whole operation process is greatly increased, and the size and the complexity of the device are increased; 2. in addition, before the elution operation of nucleic acid extraction, it is usually necessary to replace new centrifuge tubes (the waste liquid in the operations of lysis solution, binding solution and rinsing solution in the front is collected in the centrifuge tubes, and finally, when elution is performed, the centrifuge tubes are replaced to collect the final nucleic acid extraction solution), and then the extracted nucleic acid is added and mixed with the amplification system solution for isothermal amplification, thus further increasing the operation steps.
Disclosure of Invention
The invention aims to solve the technical problem of providing a full-automatic nucleic acid extraction and constant-temperature amplification and detection integrated machine aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: the utility model provides a full-automatic nucleic acid draws, constant temperature amplification detection all-in-one, includes:
the nucleic acid extraction and amplification integrated device comprises a nucleic acid extraction and amplification container, a centrifuge for realizing centrifugal rotation of the nucleic acid extraction and amplification container and a heating module for providing a heating function for the nucleic acid extraction and amplification container;
the optical detection module is used for carrying out fluorescence detection on a product obtained after nucleic acid extraction and isothermal amplification are finished in the nucleic acid extraction and amplification container;
the nucleic acid extraction and amplification container comprises a support which is rotatably arranged on a centrifugal station of the centrifuge and two centrifugal container tubes which are symmetrically arranged on the support, wherein each centrifugal container tube comprises an outer sleeve arranged on the support, a centrifuge tube inserted in the outer sleeve, a cracking tube inserted in the centrifuge tube, a plurality of pre-sealing liquid bags arranged on the inner wall of the centrifuge tube and positioned below the cracking tube, a centrifugal column arranged in the centrifuge tube and positioned below the pre-sealing liquid bags, and at least one collecting tube detachably connected to the bottom of the centrifuge tube;
the bottom of the pre-sealed liquid bag is provided with a liquid outlet, the liquid outlet is sealed through a paraffin valve, and the paraffin valve can be opened under the heating of the heating module, so that the liquid in the pre-sealed liquid bag flows out downwards through the liquid outlet.
Preferably, a heat-conducting metal ring is arranged in the liquid outlet, and paraffin is filled in the heat-conducting metal ring to form the paraffin valve; a heat conducting piece in contact with the outer wall of the heat conducting metal ring is arranged inside the inner wall of the centrifugal tube;
the heating module comprises a temperature control module and a plurality of heating sheets connected with the temperature control module, the heating sheets are arranged on the inner wall of the outer sleeve and are in contact with the heat conducting piece to heat the heat conducting metal ring, so that paraffin in the heat conducting metal ring is melted, and the paraffin valve is opened;
the plurality of pre-sealing bags comprise at least one binding solution pre-sealing bag, at least one rinsing solution pre-sealing bag and at least one eluent pre-sealing bag.
Preferably, the side wall of the centrifuge tube is provided with a mounting groove for mounting the heat conducting piece, the heat conducting piece comprises a heat conducting column in contact with the outer wall of the heat conducting metal ring and a heat conducting sheet connected to the heat conducting column, the tail end of the heat conducting sheet is bent upwards to form an outwards-protruding arc-shaped elastic sheet part, and the elastic sheet part extends out of the mounting groove and has a gap with the heat conducting sheet;
the outer wall of the heating sheet is provided with an arc-shaped groove which is used for being in matched contact with the elastic sheet part;
the heating sheets comprise a first heating sheet for heating a paraffin valve on the binding liquid pre-sealing bag, a second heating sheet for heating a paraffin valve on the rinsing liquid pre-sealing bag and a third heating sheet for heating a paraffin valve on the eluent pre-sealing bag.
Preferably, the bottom of the centrifuge tube is connected with a self-switching channel block, a self-switching channel communicated with the space below the centrifugal column is arranged in the self-switching channel block, and the self-switching channel is provided with a first outlet and a second outlet which can be switched on/off;
the collecting pipe comprises a nucleic acid sample collecting pipe connected to the first outlet and a waste liquid collecting pipe detachably connected to the second outlet, and a PCR reaction system solution for isothermal amplification is pre-filled in the nucleic acid sample collecting pipe;
the heating module further comprises a heating sleeve connected with the temperature control module, the heating sleeve is connected to the inner wall of the bottom of the centrifuge tube and surrounds the periphery of the nucleic acid sample collecting tube, and the heating sleeve is used for heating the nucleic acid sample collecting tube;
the middle of the support is connected with a hollow rotating shaft, the hollow rotating shaft is rotatably connected to a centrifugal station of the centrifugal machine, a first wire hole is formed in the hollow rotating shaft, a second wire hole communicated with the first wire hole is formed in the support, a third wire hole communicated with the second wire hole is formed in the outer sleeve, and the third wire hole is communicated to the side portion of the heating sheet and the side portion of the heating sleeve.
Preferably, the periphery of the centrifugal tube is provided with a limiting edge, one side of the limiting edge, which is close to the hollow rotating shaft, is connected with a positioning block, and the bottom of the positioning block is connected with a positioning column;
the surface of the bracket is provided with a positioning hole for inserting the positioning column, a touch switch is further arranged in the positioning hole, and after the positioning column is inserted into the positioning hole, the touch switch is switched from a disconnected state to a closed state, so that the electric connection among the temperature control module, the heating sheet and the heating sleeve is switched from a disconnected state to a connected state;
the bottom of the positioning hole is also provided with two pin holes communicated with the second wire guide hole, and the touch switch comprises a first pin and a second pin which are respectively inserted into the two pin holes, a connecting touch sheet which is rotatably connected with the upper end of the first pin, and a touch spring which is connected between the bottom surface of the connecting touch sheet and the bottom surface of the positioning hole;
when no external force acts on the connecting contact piece, the elastic force of the touch spring enables the connecting contact piece not to be in contact with the second pin, and the touch switch is in a disconnected state; when the positioning column is inserted into the positioning hole, the positioning column presses the connecting contact piece downwards, so that the connecting contact piece is contacted with the second pin, and the touch switch is switched to be in a conducting state.
Preferably, a sealing cover is rotatably connected to a pipe orifice of the cracking pipe, a hanging plate is connected to the inner wall of the cracking pipe, a cracking liquid pre-sealing bag is connected to the bottom surface of the hanging plate, at least one easy-to-tear piece is arranged on the cracking liquid pre-sealing bag, a pull rope is connected to the easy-to-tear piece, and the tail end of the pull rope is connected with the bottom of the sealing cover; when the sealing cover is completely opened, the pull rope pulls the easy-to-tear piece open to enable the cracking liquid pre-sealing bag to break and the cracking liquid in the cracking liquid pre-sealing bag flows out;
a bottom outlet of the cracking tube is connected with a porous plate, a microporous filter membrane is arranged at the upper part of the porous plate, and a pre-filter membrane is arranged above the microporous filter membrane;
the upper part of the centrifugal column is provided with a liquid distribution plate, and a plurality of liquid guide holes are densely arranged on the liquid distribution plate; a nucleic acid adsorption membrane is arranged in the centrifugal column, and a conical outlet is formed in the bottom of the centrifugal column.
Preferably, the self-switching channel comprises a switching cavity positioned in the middle, a plurality of liquid inlet holes for communicating the switching cavity with a space below the centrifugal column, a first liquid discharge channel for communicating the switching cavity with the first outlet, and a second liquid discharge channel for communicating the switching cavity with the second outlet;
a first channel port is formed at the joint of the first liquid discharge channel and the switching cavity, and a second channel port is formed at the joint of the second liquid discharge channel and the switching cavity;
the first liquid discharge channel and the second liquid discharge channel are obliquely arranged, so that the first channel opening is closer to the central axis of the centrifugal tube than the first outlet, and the second channel opening is closer to the central axis of the centrifugal tube than the second outlet;
a first spring is arranged in the switching cavity along the X direction, one end of the first spring is connected with the inner wall of the switching cavity, the other end of the first spring is connected with a sealing block which can slide in the switching cavity along the X direction, and the sealing block is used for sealing the first passage port or the second passage port;
when the centrifugal tube is kept still or rotates towards a first direction or a second direction but the rotating speed is less than Rt, the sealing block is positioned above the first passage port and completely covers the first passage port, the first passage port is closed, and the second passage port is opened;
when the centrifugal tube rotates towards a second direction and the rotating speed is not less than Rt, the first spring is stretched, the sealing block is positioned above the second passage opening and completely covers the second passage opening, the first passage opening is opened, and the second passage opening is closed;
wherein Rt is a rotating speed threshold value.
Preferably, the centrifuge comprises a frame, a motor arranged on the frame, a centrifugal rotating shaft in driving connection with an output shaft of the motor, and a centrifugal turntable connected to the centrifugal rotating shaft, wherein a plurality of bracket grooves for arranging the brackets are uniformly arranged on the centrifugal turntable at intervals, and the bracket grooves form a centrifugal station of the centrifuge;
the rack comprises a base, a first supporting disc arranged on the base and a second supporting disc arranged on the first supporting disc, wherein a plurality of detection through hole groups are arranged on the second supporting disc, and each detection through hole group comprises two detection through holes;
the optical detection module comprises two optical detection devices, and two detection heads of the two optical detection devices are respectively positioned right below two detection through holes in the same detection through hole group on the detection station;
the number and the position of the detection through hole groups are matched with those of the centrifugal container tubes, so that all the centrifugal container tubes can be subjected to optical detection successively through the two optical detection modules.
Preferably, the full-automatic nucleic acid extraction and constant temperature amplification detection all-in-one machine further comprises a shell, a cover plate arranged on the shell, a touch display screen arranged on the cover plate and a controller arranged on the centrifugal turntable and electrically connected with the touch display screen, wherein a centrifugal machine control module and a temperature control module are embedded in the controller.
Preferably, the method for carrying out nucleic acid extraction and isothermal amplification detection by the full-automatic nucleic acid extraction and isothermal amplification detection all-in-one machine comprises the following steps:
1) completely opening the sealing cover, pulling open the easy-to-tear piece on the lysate pre-sealing bag through the pull rope, so that the lysate in the lysate pre-sealing bag flows out and enters a cracking tube, and simultaneously adding a certain volume of sample into the cracking tube to cover the sealing cover;
2) inserting the centrifugal container tube into a bracket on a centrifugal station of a centrifugal machine, and aligning and inserting a positioning column on the centrifugal tube into a positioning hole of the bracket when the centrifugal container tube is inserted; completing the installation of all the centrifugal container pipes on the centrifugal station;
3) controlling the centrifuge to rotate in a positive and negative alternative way and to rotate at a speed R less than R0Working in a manner to perform sample lysis;
4) controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R meets the following requirements: r0≤R＜R1So that the lysate in the lysate passes through the microfiltration membrane and enters a centrifugal column below the microfiltration membrane, and solid impurities are intercepted by the pre-filtration membrane and the microfiltration membrane;
5) the centrifuge keeps the rotating speed R to meet the following conditions: r0≤R＜R1Rotating, controlling the first heating sheet to work through the temperature control module, heating a paraffin valve on the binding solution pre-sealing bag, opening the paraffin valve on the binding solution pre-sealing bag, allowing the inside binding solution to flow into a centrifugal column below to be mixed with lysate, and adsorbing nucleic acid through a nucleic acid adsorption membrane in the centrifugal column;
6) controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R meets the following requirements: r1R < Rt > not more than, through temperature control module control the work of second heating plate, it is right paraffin valve on the rinsing liquid bag of sealing in advance heats, makes paraffin valve on the rinsing liquid bag of sealing in advance opens, inside rinsingThe liquid flows out to rinse the centrifugal column, and then the rinsing liquid is discharged from a conical outlet below the centrifugal column;
in the processes of steps 1) -6), the sealing block is always positioned above the first channel opening and completely covers the first channel opening, the first channel opening is closed, the second channel opening is opened, so that the first outlet is always closed, the second outlet is opened, and all the waste liquid discharged from the bottom of the centrifuge tube enters the waste liquid collecting tube to be collected;
7) controlling the reverse rotation of the centrifuge, wherein the rotating speed R of the centrifuge meets the following requirements: r is not less than R2Moving the sealing block to be above the second passage opening and completely cover the second passage opening, wherein the first passage opening is opened and the second passage opening is closed; controlling the third heating sheet to work through the temperature control module, heating a paraffin valve on the eluent pre-sealing bag, opening the paraffin valve on the eluent pre-sealing bag, enabling the internal eluent to flow out to elute a centrifugal column, so that nucleic acid in the centrifugal column is separated from the nucleic acid adsorption membrane, and enabling the eluted nucleic acid and the eluent to flow into the nucleic acid sample collection pipe together for collection to obtain a nucleic acid extracting solution;
wherein R is0＜R1＜Rt＜R2；
8) Controlling the centrifuge to stop working and return to the original point, wherein all the nucleic acid sample collecting pipes are positioned right above the detection through holes at the corresponding positions;
9) the heating sleeve is controlled to work by the temperature control module, the nucleic acid sample collecting pipe is heated, and constant-temperature amplification reaction is carried out;
10) after the reaction is finished, controlling the two optical detection devices to work, simultaneously carrying out fluorescence detection on two nucleic acid sample collecting pipes in the same centrifugal container pipe right above the detection through hole at the detection station through the two optical detection devices, and then controlling the centrifugal machine to rotate in a stepping manner, so that all the centrifugal container pipes pass through the detection station one by one, and completing the fluorescence detection of all the nucleic acid sample collecting pipes.
The invention has the beneficial effects that:
the full-automatic integrated machine for nucleic acid extraction and isothermal amplification detection provided by the invention can realize automatic nucleic acid extraction, purification, isothermal amplification and detection, the lysis solution, the binding solution, the rinsing solution, the eluent and the PCR reaction system solution which are required to be used in the nucleic acid extraction process are pre-packaged in a centrifugal tube, the addition of the lysis solution is realized by the linkage of the opening action during sample addition, the paraffin valve of a pre-packaging bag is opened by a heating module through the heating action, the binding solution, the rinsing solution and the eluent can be sequentially and correspondingly released in each operation process to realize the automatic nucleic acid extraction operation, the first outlet and the second outlet which are opened/closed are automatically switched under different centrifugal rotation states by a self-switching channel, so that the waste liquid is automatically collected to a waste liquid collecting tube, the extracted nucleic acid sample is automatically collected to a nucleic acid sample collecting tube and is mixed with the PCR reaction system to realize the isothermal amplification operation, finally, optical detection is realized by the cooperation of the optical detection device and the centrifuge;
the invention avoids the defects of complex and fussy control, large equipment volume, incapability of effectively shortening the time length and the like caused by adopting a mechanical arm to carry out automatic reagent adding operation in the prior scheme, can save the time required by stopping the centrifugal operation when the mechanical arm adds the reagent, greatly simplifies the operation flow, does not need to stop a centrifugal machine to add the reagent in the nucleic acid extraction process, ensures that the whole operation process is smoother, and shortens the nucleic acid extraction time;
according to the invention, through the matching design of the self-switching channel, the second channel flowing to the waste liquid bottle can be conducted in the cracking, combining and rinsing processes of nucleic acid extraction, so that waste liquid is collected through the waste liquid bottle; the first channel flowing to the nucleic acid sample bottle is conducted in the elution process of nucleic acid extraction, and the required nucleic acid extracting solution is collected, so that a liquid storage bottle for collecting reagents does not need to be replaced in the whole detection process, and the operation steps are further simplified;
the nucleic acid sample extracted in the invention can be directly mixed with the PCR reaction system solution for isothermal amplification, the nucleic acid sample does not need to be transferred, and further, the amplified product is directly optically detected and does not need to be transferred, thereby further simplifying the process and improving the detection efficiency.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the fully automatic nucleic acid extraction and isothermal amplification detection all-in-one machine of the present invention;
FIG. 2 is a schematic diagram of the internal structure of the fully automatic nucleic acid extraction and isothermal amplification detection all-in-one machine of the present invention;
FIG. 3 is a schematic diagram of the structure of a nucleic acid extraction and amplification vessel according to the present invention;
FIG. 4 is a schematic view showing the internal structure of the nucleic acid extraction and amplification vessel according to the present invention;
FIG. 5 is a schematic view of the internal structure of the stent of the present invention;
FIG. 6 is an enlarged partial view of FIG. 4A;
FIG. 7 is a schematic view of the structure of the positioning post of the present invention cooperating with the touch switch;
FIG. 8 is a schematic diagram of the structure of the centrifuge vessel tube of the present invention;
FIG. 9 is a schematic structural view of a cracking tube of the present invention;
FIG. 10 is a schematic view of the seal cap of the centrifuge container tube of the present invention in an open configuration;
FIG. 11 is a schematic view of a centrifugal column according to the present invention;
FIG. 12 is a schematic view of the configuration of the self-switching channel block and collection tube of the present invention in cooperation;
FIG. 13 is a diagram illustrating a self-switching channel block according to an embodiment of the present invention;
fig. 14 is a structural diagram illustrating a second outlet closed state in the self-switching channel block according to the present invention.
Description of reference numerals:
1-nucleic acid extraction and amplification integrated device;
2-nucleic acid extraction and amplification container; 20-a support; 21-centrifuge vessel tube; 22 — an outer sleeve; 23-centrifuging the tube; 24-a cracking tube; 25-pre-sealing the liquid bag; 26-centrifugal column; 27-a collection pipe;
200-hollow rotating shaft; 201-first wire guides; 202-a second wire guide; 203-positioning holes; 204-touch switch; 205-pin holes; 2040-first lead; 2041 — second lead; 2042-connecting contact piece; 2043 — a touch spring;
220-third wire guides;
230-a limiting edge; 231-a positioning block; 232-positioning column; 233, mounting grooves;
240-sealing cover; 241-hanging plate; 242-pre-sealing the bag with the lysate; 243-easy-tear piece; 244-pulling a rope; 245-suspension loop; 246-perforated plate; 247-microfiltration membranes; 248-pre-filtration membrane;
250-a liquid outlet; 251-paraffin valve; 252 — a thermally conductive metal ring; 253-a thermally conductive member; 254-pre-sealing the bag with the binding liquid; 255-pre-sealing the bag by using rinsing liquid; 256-pre-sealing bag of eluent; 2530-heat conducting columns; 2531-heat conducting fins; 2532-tab portion;
260-liquid distribution plate; 261-nucleic acid adsorption membrane; 262-a conical outlet; 263-liquid distribution hole;
270-a nucleic acid sample collection tube; 271-waste liquid collecting pipe; 272-heat conducting sleeve;
3-a centrifuge; 30-a centrifugal station; 31-a frame; 32, a motor; 33-centrifugal rotating shaft; 34-a centrifugal turntable; 35-bracket groove; 310-a base; 311 — a first support disc; 312 — a second support disk; 313 — a set of detection vias; 314 — detect the via;
4-heating the module; 40-heating plates; 41-heating jacket; 400-arc groove; 401 — a first heating plate; 402-a second heating plate; 403 — third heating plate;
5-self-switching channel block; 50-self-switching channel; 51-a first spring; 52-a sealing block; 53-top post; 500-liquid inlet hole; 501, switching a cavity; 502 — first passage port; 503-second access; 504-first drain channel; 505 — second drainage channel 505; 506 — a first outlet; 507 — a second outlet; 530-hollow column; 531 — second spring; 532-heading the ball;
6-optical detection device; 60-a detection head;
7-a housing; 70-a cover plate; 71-touch display screen; 72-controller.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
As shown in fig. 1 to 12, this embodiment provides a full-automatic nucleic acid extraction and isothermal amplification detection all-in-one machine, which includes:
the nucleic acid extraction and amplification integrated device 1 comprises a nucleic acid extraction and amplification container 2, a centrifuge 3 for realizing centrifugal rotation of the nucleic acid extraction and amplification container 2 and a heating module 4 for providing a heating function for the nucleic acid extraction and amplification container 2;
the optical detection module is used for carrying out fluorescence detection on a product obtained after nucleic acid extraction and isothermal amplification are completed in the nucleic acid extraction and amplification container 2;
the nucleic acid extraction and amplification container 2 comprises a support 20 which is rotatably arranged on a centrifugal station 30 of the centrifuge 3 and two centrifugal container tubes 21 which are symmetrically arranged on the support 20, wherein each centrifugal container tube 21 comprises an outer sleeve 22 arranged on the support 20, a centrifuge tube 23 inserted in the outer sleeve 22, a lysis tube 24 inserted in the centrifuge tube 23, a plurality of pre-sealed liquid bags 25 which are arranged on the inner wall of the centrifuge tube 23 and are positioned below the lysis tube 24, a centrifugal column 26 which is arranged in the centrifuge tube 23 and is positioned below the pre-sealed liquid bags 25, and at least one collecting tube 27 which is detachably connected to the bottom of the centrifuge tube 23;
the bottom of the pre-sealing liquid bag 25 is provided with a liquid outlet 250, the liquid outlet 250 is sealed by a paraffin valve 251, and the paraffin valve 251 can be opened under the heating of the heating module 4, so that the liquid in the pre-sealing liquid bag 25 flows out downwards through the liquid outlet 250.
In a preferred embodiment, the centrifuge 3 includes a frame 31, a motor 32 disposed on the frame 31, a centrifugal rotating shaft 33 drivingly connected to an output shaft of the motor 32, and a centrifugal rotating disk 34 connected to the centrifugal rotating shaft 33, wherein a plurality of bracket slots 35 for disposing the brackets 20 are disposed on the centrifugal rotating disk 34 at regular intervals, and the bracket slots 35 form a centrifugal station 30 of the centrifuge 3;
the rack 31 comprises a base 310, a first supporting disk 311 arranged on the base 310 and a second supporting disk 312 arranged on the first supporting disk 311, wherein the second supporting disk 312 is provided with a plurality of detection through hole groups 313, and each detection through hole group 313 comprises two detection through holes 314;
the optical detection module comprises two optical detection devices 6, and two detection heads 60 of the two optical detection devices 6 are respectively positioned right below two detection through holes 314 in the same detection through hole group 313 on the detection station;
the number and position of the detection via groups 313 are matched to the centrifuge vessel tubes 21, so that all centrifuge vessel tubes 21 can be optically detected one by two optical detection modules. That is, the fluorescence detection of the two centrifuge vessel tubes 21 can be realized by the two optical detection devices 6 at each time, then the centrifuge 3 rotates one position, the two centrifuge vessel tubes 21 on the next bracket 20 rotate to the positions above the two optical detection devices 6, then the optical detection is performed, and the detection of the centrifuge vessel tubes 21 on all the brackets 20 can be realized in sequence by the cooperation of the centrifuge 3 and the two optical detection devices 6.
In a preferred embodiment, the full-automatic nucleic acid extraction and isothermal amplification detection all-in-one machine further comprises a housing 7, a cover plate 70 arranged on the housing 7, a touch display screen 71 arranged on the cover plate 70, a controller 72 arranged on the centrifugal turntable 34 and electrically connected with the touch display screen 71, and a power supply arranged on the centrifugal turntable 34 and connected with the controller 72, wherein a central control module, a centrifuge 3 control module, an optical detection control module and a temperature control module are embedded in the controller 72.
Wherein, the optical detection device 6 can adopt conventional products for realizing fluorescence detection. For example, in one embodiment, the device includes a laser light source, an optical filter, an objective lens, a detector, etc., wherein laser light emitted from the laser light source passes through the objective lens and then irradiates the sample in the centrifuge container tube 21, and fluorescence generated by excitation of the sample passes through the objective lens and the optical filter and then reaches the detector for fluorescence detection. The central control module obtains the data of the detector, processes the data to obtain a fluorescence detection result, and transmits the fluorescence detection result to the touch display screen 71 for displaying, and the optical detection control module is used for controlling the operation of the optical detection device 6.
The rechargeable storage battery can be used as a power supply to supply power, and the rechargeable storage battery can also be externally connected with the power supply to supply power. The touch display screen 71 can be used for displaying the final detection result, and can also be used as an input interface for inputting corresponding control parameters, such as centrifugal rotation speed, temperature control, and the like. Wherein each centrifugation container is assigned a corresponding number for distinction.
In this embodiment, a heat-conducting metal ring 252 is disposed in the liquid outlet 250, and paraffin is filled in the heat-conducting metal ring 252 to form a paraffin valve 251; a heat-conducting piece 253 which is contacted with the outer wall of the heat-conducting metal ring 252 is arranged inside the inner wall of the centrifugal tube 23;
the heating module 4 comprises a temperature control module and a plurality of heating sheets 40 connected with the temperature control module, wherein the heating sheets 40 are arranged on the inner wall of the outer sleeve 22 and are in contact with the heat-conducting piece 253 to heat the heat-conducting metal ring 252, so that paraffin in the heat-conducting metal ring 252 is melted, and the paraffin valve 251 is opened;
the plurality of pre-seal fluid bags 25 includes at least one binding fluid pre-seal bag 254, at least one rinse fluid pre-seal bag 255, and at least one eluent pre-seal bag 256. In this embodiment, a binding liquid pre-sealing bag 254, a rinsing liquid pre-sealing bag 255, and an eluent pre-sealing bag 256 are taken as an example for illustration, one binding liquid pre-sealing bag 254, one rinsing liquid pre-sealing bag 255, and one eluent pre-sealing bag 256 are uniformly connected to the inner wall of the centrifuge tube 23 at intervals, and paraffin is melted by controlling the operation of the heating sheet 40, so that the paraffin valve 251 on any one pre-sealing bag can be independently opened, and release of the solution in the pre-sealing bag as required is realized, so that the centrifuge 3 does not need to be stopped in the process of nucleic acid extraction, and reagents are automatically added manually or through components such as a mechanical arm. In a preferred embodiment, the temperature required for opening the paraffin valve 251 is 58-63 deg.C, and the paraffin valve 251 is opened by heating the heating plate 40 for several seconds.
Referring to fig. 4 to 6, in the embodiment, a mounting groove 233 for mounting a heat conducting member 253 is formed on a side wall of the centrifugal tube 23, the heat conducting member 253 includes a heat conducting column 2530 contacting with an outer wall of the heat conducting metal ring 252 and a heat conducting sheet 2531 connected to the heat conducting column 2530, a terminal of the heat conducting sheet 2531 is bent upwards to form an arc-shaped convex elastic sheet portion 2532, and the elastic sheet portion 2532 extends out of the mounting groove 233 and has a gap with the heat conducting sheet 2531; the outer wall of the heating plate 40 has an arc-shaped groove 400 for fitting contact with the spring portion 2532.
Referring to fig. 6, the heat provided by the heating plate 40 is transferred to the heat-conducting metal ring 252 through the elastic sheet portion 2532, the heat-conducting sheet 2531 and the heat-conducting column 2530, and paraffin is wrapped by the heat-conducting metal ring 252, so that the paraffin can be efficiently melted by the heat of the heat-conducting metal ring 252, and the paraffin valve 251 can be rapidly opened.
Conducting strip 2531 and shell fragment portion 2532's structural design, make shell fragment portion 2532 have certain elastic force effect, when centrifuging tube 23 inserts in the overcoat pipe, shell fragment portion 2532 is earlier by the extrusion and is close to conducting strip 2531, when inserting the back that targets in place, shell fragment portion 2532 just in time is in arc recess 400 lateral part of heating plate 40, in shell fragment portion 2532 pushes up arc recess 400, can guarantee on the one hand that shell fragment portion 2532 has good contact with heating plate 40, guarantee heat conduction efficiency, on the other hand pressure effect between the two also can play the fixed action to centrifuging tube 23 to a certain extent, make centrifuging tube 23 can more stable setting in the overcoat pipe.
Wherein, the plurality of heating sheets 40 comprise a first heating sheet 401 for heating the paraffin valve 251 on the binding liquid pre-sealing bag 254, a second heating sheet 402 for heating the paraffin valve 251 on the rinsing liquid pre-sealing bag 255 and a third heating sheet 403 for heating the paraffin valve 251 on the eluent pre-sealing bag 256. The first heating plate 401, the second heating plate 402 and the third heating plate 403 are independently controlled by the temperature control module, so that the paraffin valves 251 on the binding liquid pre-sealing bag 254, the rinsing liquid pre-sealing bag 255 and the eluent pre-sealing bag 256 can be opened as required, and the binding liquid, the rinsing liquid and the eluent are sequentially released according to the requirements of the set steps so as to meet the requirements of nucleic acid extraction operation.
In this embodiment, the bottom of the centrifugal tube 23 is connected with a self-switching channel 50 block 5, the self-switching channel 50 block 5 has a self-switching channel 50 therein, which is communicated with the space below the centrifugal column 26, and the self-switching channel 50 has a first outlet 506 and a second outlet 507 which can be switched on/off;
the collection pipe 27 comprises a nucleic acid sample collection pipe 270 connected with the first outlet 506 and a waste liquid collection pipe 271 detachably connected with the second outlet 507, and a PCR reaction system solution for isothermal amplification is pre-filled in the nucleic acid sample collection pipe 270;
the heating module 4 further comprises a heating sleeve 41 connected with the temperature control module, the heating sleeve 41 is connected to the inner wall of the bottom of the centrifuge tube 23 and surrounds the periphery of the nucleic acid sample collection tube 270, and is used for heating the nucleic acid sample collection tube 270 to perform isothermal amplification reaction; in a preferred embodiment, the nucleic acid sample collection tube 270 is externally wrapped with a thermally conductive sheath 272 for increased heat transfer efficiency.
The middle part of support 20 is connected with hollow rotating shaft 200, and hollow rotating shaft 200 rotatable coupling is on centrifuge 3's centrifugation station 30, and support 20 connects on centrifuge 3's centrifugal carousel 34 through hollow rotating shaft 200, and when not rotating, centrifugal container pipe 21 is vertical state, and when centrifugal carousel 34 rotated, support 20 rotated relative centrifugal carousel 34 under the effect of centrifugal force, and centrifugal container pipe 21 becomes and is close the horizontality.
The hollow rotating shaft 200 has a first wire guiding hole 201 inside, the holder 20 has a second wire guiding hole 202 inside, the outer sleeve 22 has a third wire guiding hole 220 communicating with the second wire guiding hole 202, and the third wire guiding hole 220 communicates with the side of the heating plate 40 and the side of the heating jacket 41. The first wire guide 201, the second wire guide 202 and the third wire guide 220 are used for connecting the wiring step of wires, which connect the heating chip 40 and the heating jacket 41 through the wires, to supply the external power to the heating chip 40 and the heating jacket 41 without interfering with the rotation of the centrifuge 3. For example, in one embodiment, the power supply and the temperature control module are disposed on the centrifugal turntable 34 of the centrifuge 3, and the centrifugal turntable 34 is provided with a wire slot/hole, the temperature control module is electrically connected to the power supply through a wire, the temperature control module is further led out of the wire, enters from the first wire hole 201 in the hollow rotating shaft 200, is respectively connected to the heating jacket 41 and each heating sheet 40 through the second wire hole 202 and the third wire hole 220, and the heating jacket 41 and each heating sheet 40 can be controlled by the temperature control module to respectively and independently operate. Since the power supply and the temperature control module are both disposed on the centrifugal turntable 34 and rotate together with the same, the electrical connections between the power supply, the temperature control module and the heating jackets 41 and the respective heating sheets 40 are not interfered or affected by the centrifugal rotation.
In this embodiment, the periphery of centrifuging tube 23 is provided with spacing border 230, and centrifuging tube 23 inserts the outer tube and targets in place the back, and spacing border 230 card carries out the axial spacing on the support 20 surface.
One side of the limiting edge 230 close to the hollow rotating shaft 200 is connected with a positioning block 231, and the bottom of the positioning block 231 is connected with a positioning column 232; the surface of the bracket 20 is provided with a positioning hole 203 for inserting the positioning column 232, a touch switch 204 is further arranged in the positioning hole 203, and after the positioning column 232 is inserted into the positioning hole 203, the touch switch 204 is switched from a disconnected state to a closed state, so that the electric connection between the temperature control module and the heating sheet 40 and between the temperature control module and the heating sleeve 41 is switched from a disconnected state to a connected state;
referring to fig. 5 and 7, in a preferred embodiment, the bottom of the positioning hole 203 is further opened with two pin holes 205 communicating with the second wire hole 202, and the tact switch 204 includes a first pin 2040 and a second pin 2041 respectively inserted into the two pin holes 205, a connecting contact 2042 rotatably connected to the upper end of the first pin 2040, and a tact spring 2043 connected between the bottom surface of the connecting contact 2042 and the bottom surface of the positioning hole 203;
when no external force acts on the connecting contact 2042, the elastic force of the touch spring 2043 prevents the connecting contact 2042 from contacting the second pin 2041, and the touch switch 204 is in an off state; when the positioning post 232 is inserted into the positioning hole 203, the positioning post 232 presses the connecting contact 2042 downward, so that the connecting contact 2042 contacts the second pin 2041, and the trigger switch 204 is switched to the on state. The first pin 2040 and the second pin 2041 are connected to the wire loops of the heating plate 40 and the heating jacket 41, and when the connecting contact plate 2042 is in contact with the second pin 2041, the first pin 2040 and the second pin 2041 are communicated, and the wire loops are conducted; otherwise the wire loop is broken.
On one hand, the cooperation of the positioning column 232 and the positioning hole 203 can position the target position of the centrifugal tube 23 inserted into the outer sleeve, play a role in indicating the installation position of the centrifugal tube 23, and prevent the centrifugal tube 23 from rotating relative to the outer sleeve; on the other hand, the cooperation of the positioning column 232 and the touch switch 204 can also indicate whether the centrifugal container tube 21 is inserted into the outer sleeve, and further perform general control on whether heat is provided to the heating sheet 40 and the heating sleeve 41 in the sleeve, and only when the centrifugal container tube 21 is installed in the outer sleeve in place, the electric connection loop of the heating sheet 40 and the heating sleeve 41 is conducted, so that the heating sheet 40 and the heating sleeve 41 can be controlled to work as required, and therefore, electric energy can be saved, and a complex control method or mechanism is not needed.
Referring to fig. 8-10, in this embodiment, a sealing cover 240 is rotatably connected to a nozzle of the cracking tube 24, a hanging plate 241 is connected to an inner wall of the cracking tube 24, a cracking solution pre-sealing bag 242 is connected to a bottom surface of the hanging plate 241, at least one easy-to-tear piece 243 is arranged on the cracking solution pre-sealing bag 242, a pulling rope 244 is connected to the easy-to-tear piece 243, and a tail end of the pulling rope 244 is connected to a bottom of the sealing cover 240; when the sealing lid 240 is fully opened, the pull cord 244 pulls the tear tab 243 open to break the lysate pre-sealed bag 242 and allow the lysate to flow out of the inner portion.
In a preferred embodiment, the bottom of the sealing cover 240 is provided with a hanging ring 245 for connecting the pulling rope 244, when the cracking tube 24 is produced and packaged, after the cracking solution pre-sealing bag 242 in which the cracking solution is packaged is arranged on the hanging plate 241, the sealing cover 240 is half-opened, so that the tail end of the pulling rope 244 can be fixedly connected to the hanging ring 245, and then the sealing cover 240 is buckled and sealed, as shown in fig. 8 and 9. The length of the pull rope 244 is suitable, and the length of the pull rope can be approximately equal to the linear distance between the hanging ring 245 and the easy-to-tear piece 243 closest to the hanging ring 245 when the sealing cover 240 is half opened, so that when the sealing cover 240 is half opened, all the easy-to-tear pieces 243 cannot be torn off, and when the sealing cover 240 is completely opened, all the easy-to-tear pieces 243 can be torn off to release the lysate in the lysate pre-sealed bag 242, referring to fig. 10; through the linkage setting of sealed lid 240 and easy tear piece 243 for when this centrifuging tube 23 used, can add the lysate by automatic when opening sealed lid 240 and adding the sample completely, can simplify the step of a adding the lysate. After the sample is added, the sealing cover 240 is buckled to wait for the next operation.
In this embodiment, a porous plate 246 is connected to the bottom outlet of the cracking tube 24, a microporous membrane 247 is disposed on the upper portion of the porous plate 246, and a pre-filtration membrane 248 is disposed above the microporous membrane 247.
The pre-filtering membrane 248 is arranged above and is used for filtering out some large-particle impurities generated after the sample is cracked, and the blocking of the microporous filtering membrane 247 below can be prevented. The microfiltration membrane 247 is a hydrophobic membrane and is provided with a micropore array, so that when no external force acts, liquid above the microfiltration membrane 247 cannot or cannot basically flow into the lower space through the microfiltration membrane 247, when certain external force (such as centrifugal force) is received, the liquid above the microfiltration membrane 247 can flow into the lower space through the microfiltration membrane 247, most of solid impurities are intercepted above the microfiltration membrane 247, and a space for fully mixing a sample and a lysate can be formed above the microfiltration membrane 247. For example, in an alternative embodiment, the microporous filter membrane 247 may be a polytetrafluoroethylene filter membrane, a polypropylene filter membrane, a polyethersulfone filter membrane, or the like, and the size of the micropores in the microporous array thereon is 0.05um to 100 um. When it is understood that the smaller the pore size is, the more external force is required for the liquid above the microporous filter membrane 247 to pass through the microporous filter membrane 247, and the larger the centrifugal rotation speed is required to be provided corresponding to the centrifugal force as the external force, so that the centrifugal rotation speed (for example, R) not less than a certain value can be satisfied by setting the appropriate pore size0) The lower liquid may pass through the microfiltration membrane 247, but below this centrifugation speed the liquid may not pass or substantially not pass through the microfiltration membrane 247.
Referring to fig. 11, in the present embodiment, a liquid distribution plate 260 is disposed on the upper portion of the centrifugal column 26, and a plurality of liquid distribution holes 263 are densely disposed on the liquid distribution plate 260; a nucleic acid adsorption membrane 261 is arranged in the centrifugal column 26, and a conical outlet 262 is arranged at the bottom of the centrifugal column 26. The liquid distribution plate 260 collects the reagents (binding solution, rinsing solution, eluent, etc.) released in the upper pre-sealed bag, and then the reagents are uniformly distributed through a plurality of liquid guide holes and flow into the nucleic acid adsorption film 261 below. The same nucleic acid adsorption membrane 261 may be usedAdopting a material having affinity or adsorption capacity for nucleic acid, which has a pore array having a pore size of 0.02-50 um, the pore size of the pore array being set so as to be at a centrifugal rotation speed (e.g., R) of not less than a certain value1) The lower liquid may pass through the microfiltration membrane 247, but below this centrifugation speed the liquid may not pass or substantially not pass through the microfiltration membrane 247.
The reagents in the lysate pre-sealing bag 242, the binding solution pre-sealing bag 254, the rinsing solution pre-sealing bag 255 and the eluent pre-sealing bag 256 are all prepared from the conventional lysate, the binding solution, the rinsing solution and the eluent which are respectively used for nucleic acid extraction at each stage, the reagents are respectively pre-sealed and arranged at corresponding positions in the centrifuge tube 23, the amounts of the reagents are matched according to a preset certain sample adding amount, the range of the sample adding amount can be marked on the outer wall of the centrifuge tube 23, or the sample adding amount range can be explained in other modes. For example, in one embodiment, the amount of the standard sample added to the sealing cap 240 of the centrifuge tube 23 is in the range of 1-5mL, and the pre-sealed amount of each reagent is pre-filled accordingly; therefore, when in use, only a certain amount of sample is required to be added according to requirements, and the rest of the reagent amount can be matched with the sample, and is not required to be added.
Wherein, a sufficient amount of rinsing liquid is packaged by the rinsing liquid pre-sealing bag 255, so that the requirement can be met by one-time rinsing. It will be appreciated that a plurality of rinse solution pre-bags 255 may be provided to perform multiple rinses, each time releasing a quantity of rinse solution in the rinse solution pre-bags 255 by controlling the temperature of the paraffin valve 251. As a preferred scheme, only one rinsing is performed, thereby simplifying the steps and shortening the time.
In this embodiment, the automatic distribution and collection function of the waste liquid and the nucleic acid liquid in the nucleic acid extraction process is realized by the self-switching channel 50, and the need of replacing the collection tube 27 in the nucleic acid extraction process as in the conventional scheme can be avoided.
Referring to fig. 12, in the present embodiment, the self-switching channel 50 includes a switching cavity 501 located in the middle, a plurality of liquid inlet holes 500 communicating the switching cavity 501 with the space below the centrifugal column 26, a first liquid drainage channel 504 communicating the switching cavity 501 with a first outlet 506, and a second liquid drainage channel 505 communicating the switching cavity 501 with a second outlet 507;
a first channel opening 502 is formed at the joint of the first drainage channel 504 and the switching cavity 501, and a second channel opening 503 is formed at the joint of the second drainage channel 505 and the switching cavity 501;
the first drainage channel 504 and the second drainage channel 505 are both arranged obliquely such that the first channel opening 502 is closer to the central axis of the centrifuge tube 23 than the first outlet 506, and the second channel opening 503 is closer to the central axis of the centrifuge tube 23 than the second outlet 507;
a first spring 51 is arranged in the switching cavity 501 along the X direction, one end of the first spring 51 is connected with the inner wall of the switching cavity 501, the other end of the first spring 51 is connected with a sealing block 52 which can slide in the switching cavity 501 along the X direction, and the sealing block 52 is used for sealing the first passage opening 502 or the second passage opening 503;
when the centrifugal tube 23 is still or rotates in the first direction or rotates in the second direction but the rotation speed is less than Rt, the sealing block 52 is located above the first passage opening 502 and completely covers the first passage opening 502, the first passage opening 502 is closed, and the second passage opening 503 is opened;
when the centrifugal tube 23 rotates in the second direction and the rotation speed is not less than Rt, the first spring 51 is stretched, the sealing block 52 is located above the second passage opening 503 and completely covers the second passage opening 503, the first passage opening 502 is opened, and the second passage opening 503 is closed;
wherein Rt is a rotating speed threshold value.
Referring to fig. 8, 12 and 14, the following describes in more detail the automatic switching process of the sealing block 52 and the first spring 51 to open/close the first and second ports 502 and 503 at different centrifugal rotation speeds. In the figure, the arrow indicates the centrifugal rotation direction, and O indicates the rotation center.
Referring to fig. 8, when the centrifugal tube 23 rotates centrifugally, the sealing block 52 moves left and right under the action of centrifugal force, and the moving direction depends on the rotating direction, for example, if the centrifugal tube rotates clockwise, the sealing block 52 moves left, and if the centrifugal tube rotates counterclockwise, the sealing block 52 moves right; while the sealing block 52 is fixed by the first spring 51, the movement of the sealing block 52 depends on the centrifugal action and the elastic force to which the sealing block 52 is subjectedThe resultant of the actions. When rotating clockwise, as shown in fig. 8, the sealing block 52 moves leftwards to compress the first spring 51, and the lengths of the first spring 51 and the sealing block 52 are designed such that when the first spring 51 is compressed to the shortest, the lower part of the sealing block 52 still completely covers the first passage opening 502, so that when rotating clockwise, the first passage opening 502 is always closed. When rotating counterclockwise, the sealing block 52 moves rightwards to stretch the first spring 51, the sealing block 52 moves rightwards gradually as the rotating speed increases, when the rotating speed reaches Rt, the sealing block 52 starts to be separated from the first passage port 502, and the first passage port 502 starts to be opened; as the speed continues to increase, when increasing to R2At this time, the sealing block 52 is completely separated from the first opening 502 and completely covers the second opening 503, so that the first opening 502 is opened and the second opening 503 is closed, as shown in fig. 14. Due to the limitation of the inner wall at the right side of the switching cavity 501 on the sealing block 52, when the rotating shaft rotates in the anticlockwise direction, the rotating speed R is larger than or equal to R2Then, the sealing block 52 cannot move any further because it is blocked by the inner wall on the right side of the switching cavity 501, and the second opening 503 still remains closed.
Referring to fig. 13, in a preferred embodiment, a top pillar 53 is further disposed above the sealing block 52 along the Z direction, and the top pillar 53 includes a hollow pillar 530 connected to the sealing block 52, a second spring 531 connected inside the hollow pillar 530, and a top ball 532 connected to an upper end of the second spring 531 and movably disposed inside the hollow pillar 530; wherein the top ball 532 has a diameter greater than the diameter of the top opening of the hollow cylinder 530 such that the top ball 532 can rotate without being disengaged from the inside of the hollow cylinder 530.
The knob 532 contacts with the upper inner wall of the switching cavity 501, and the liquid inlet hole 500 above the switching cavity 501 avoids the movement track of the knob 532, that is, the knob 532 does not have the liquid inlet hole 500 at the contact part with the upper inner wall of the switching cavity 501 during the movement process, so that the knob 532 is prevented from being clamped into the liquid inlet hole 500.
When the sealing block 52 moves, the jacking ball 532 is in rolling contact with the upper inner wall of the switching cavity 501, the ground of the sealing block 52 can be kept in tight contact with the bottom inner wall of the switching cavity 501 through jacking action, and meanwhile, the sealing block 52 can still move smoothly under the centrifugal action through rolling contact.
In a further preferred embodiment, the sealing layer (e.g. teflon layer) is disposed on the bottom surface of the sealing block 52, and the surface is flat and smooth, and the sealing layer (e.g. teflon layer) may also be disposed on the inner wall of the bottom of the switching cavity 501 to ensure that the surface is flat and smooth, so that the sealing block 52 can move smoothly and achieve a good sealing when moving to a proper position.
Through the matching design of the structure of the self-switching channel 50, a second channel flowing to a waste liquid bottle can be conducted in the processes of cracking, combining and rinsing of nucleic acid extraction, so that waste liquid is collected through the waste liquid bottle; the first channel flowing to the nucleic acid sample bottle is conducted in the elution process of nucleic acid extraction, and the required nucleic acid extracting solution is collected, so that the liquid storage bottle for collecting the reagent is not required to be replaced in the nucleic acid extraction process; this is further illustrated in example 2 below.
Example 2
In this embodiment, a method for performing nucleic acid extraction and isothermal amplification detection by using the full-automatic integrated machine for nucleic acid extraction and isothermal amplification detection of embodiment 1 is provided, which includes the following steps:
1) completely opening the sealing cover 240, pulling open the easily-torn piece 243 on the lysate pre-sealing bag 242 through the pull rope 244, so that the lysate in the lysate pre-sealing bag 242 flows out and enters the cracking tube 24, meanwhile, a certain volume of sample is added into the cracking tube 24, and the sealing cover 240 is covered; at this time, the centrifugal column 26 tube can be manually shaken to preliminarily mix the lysate and the sample;
2) inserting the centrifugal container tube 21 into the bracket 20 on the centrifugal station 30 of the centrifuge 3, aligning the positioning column 232 on the centrifugal tube 23 and inserting into the positioning hole 203 of the bracket 20 when inserting, and conducting the touch switch 204 through the positioning column 232 so as to communicate with the heating loop; the installation of all the centrifuge vessel tubes 21 on the centrifuge station 30 is completed in sequence;
3) controlling the centrifuge 3 to rotate alternately in the positive and negative directions and to rotate at a speed R less than R0Working in a manner to perform sample lysis; in the process, the lysate and the sample do not substantially pass through the microporous filter membrane 247 due to the low rotation speed, and thus stay in the lysis tube 24 and are provided by the cavity above the microporous filter membrane 247A mixing space for the lysate and the sample;
4) controlling the centrifuge 3 to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r0≤R＜R1So that the lysate in the lysate passes through the microporous filter membrane 247 and enters the centrifugal column 26 below, and the solid impurities are intercepted by the pre-filtering membrane 248 and the microporous filter membrane 247; due to the increase of the rotating speed, the lysate can pass through the micropore array on the micropore filter membrane 247;
5) the centrifuge 3 keeps the rotating speed R satisfying: r0≤R＜R1Rotating, controlling the first heating plate 401 to work through the temperature control module, heating the paraffin valve 251 on the binding solution pre-sealing bag 254, opening the paraffin valve 251 on the binding solution pre-sealing bag 254, allowing the inside binding solution to flow into the centrifugal column 26 below to be mixed with the lysate, and adsorbing the nucleic acid through the nucleic acid adsorption film 261 in the centrifugal column 26; in this process, since the size of the micro-pore array on the nucleic acid adsorption film 261 is set, the binding solution, the sample, and the like on the nucleic acid adsorption film 261 do not substantially pass through the nucleic acid adsorption film 261 at this rotation speed, thereby providing space and time for mixing the binding solution and the sample, and nucleic acid is adsorbed by the nucleic acid adsorption film 261 under the action of the binding solution;
6) controlling the centrifuge 3 to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: r1R is less than or equal to Rt, then the second heating sheet 402 is controlled to work through the temperature control module, the paraffin valve 251 on the rinsing liquid pre-sealing bag 255 is heated, the paraffin valve 251 on the rinsing liquid pre-sealing bag 255 is opened, the rinsing liquid in the rinsing liquid pre-sealing bag flows out to rinse the centrifugal column 26, and then the rinsing liquid is discharged from the conical outlet 262 below the centrifugal column 26; at this rotation speed, the liquid can pass through the nucleic acid adsorption film 261, so that the rinsing liquid and the washed impurities pass through the nucleic acid adsorption film 261 into the waste liquid collection pipe 271;
in the processes of steps 1) -6), the sealing block 52 is always positioned above the first channel opening 502 and completely covers the first channel opening 502, the first channel opening 502 is closed, the second channel opening 503 is opened, so that the first outlet 506 is always closed, the second outlet 507 is opened, and all the waste liquid discharged from the bottom of the centrifugal tube 23 enters the waste liquid collecting tube 271 for collection;
7) the centrifugal machine 3 is controlled to rotate reversely,and the rotation speed R of the centrifuge 3 satisfies: r is not less than R2So that the sealing block 52 moves over the second opening 503 and completely covers the second opening 503, the first opening 502 is open, and the second opening 503 is closed; meanwhile, the temperature control module controls the third heating plate 403 to work, the paraffin valve 251 on the eluent pre-sealing bag 256 is heated, the paraffin valve 251 on the eluent pre-sealing bag 256 is opened, the internal eluent flows out to elute the centrifugal column 26, nucleic acid in the centrifugal column 26 is separated from the nucleic acid adsorption film 261, and the eluted nucleic acid and the eluent flow into the nucleic acid sample collection pipe 270 together to be collected, so that a nucleic acid extracting solution is obtained, and nucleic acid extraction is completed;
wherein R is0＜R1＜Rt＜R2；
8) Controlling the centrifuge 3 to stop working and return to the origin, wherein all the nucleic acid sample collection tubes 270 are located right above the detection through holes 314 at the corresponding positions, and the two nucleic acid sample collection tubes 270 on the first bracket 20 are respectively located right above the two optical detection devices 6;
9) the heating sleeve 41 is controlled to work by the temperature control module, the nucleic acid sample collecting pipe 270 is heated, and constant-temperature amplification reaction is carried out;
10) after the reaction is finished, the two optical detection devices 6 are controlled to work, the two nucleic acid sample collecting pipes 270 in the same centrifugal container pipe 21 right above the detection through hole 314 at the detection station are simultaneously subjected to fluorescence detection through the two optical detection devices 6, and then the centrifuge 3 is controlled to rotate in a stepping mode, so that all the centrifugal container pipes 21 pass through the detection station one by one, the fluorescence detection of all the nucleic acid sample collecting pipes 270 is finished, and the display is carried out through the touch display screen 71.
Example 3
This example further illustrates the present invention by taking the full-automatic integrated machine for nucleic acid extraction and isothermal amplification detection of example 1 as an example for performing loop-mediated isothermal amplification detection.
The method for carrying out nucleic acid extraction and isothermal amplification detection by the full-automatic nucleic acid extraction and isothermal amplification detection all-in-one machine in the embodiment comprises the following steps:
1) completely opening the sealing cover 240, pulling open the easily-torn piece 243 on the lysate pre-sealing bag 242 through the pull rope 244, so that the lysate in the lysate pre-sealing bag 242 flows out and enters the lysis tube 24, meanwhile, 3mL of sample is added into the lysis tube 24, and the sealing cover 240 is well covered; at this time, the centrifugal column 26 tube can be manually shaken to preliminarily mix the lysate and the sample;
2) inserting the centrifugal container tube 21 into the bracket 20 on the centrifugal station 30 of the centrifuge 3, aligning the positioning column 232 on the centrifugal tube 23 and inserting into the positioning hole 203 of the bracket 20 when inserting, and conducting the touch switch 204 through the positioning column 232 so as to communicate with the heating loop; the installation of all the centrifuge vessel tubes 21 on the centrifuge station 30 is completed in sequence;
3) controlling the centrifuge 3 to work for 10s in a mode of forward and reverse alternate rotation and at a rotating speed R of 650R/min to perform sample cracking; in the process, as the rotating speed is low, the lysate and the sample basically do not pass through the microporous filter membrane 247, so that the lysate and the sample stay in the lysis tube 24, and a mixing space of the lysate and the sample is provided by a cavity above the microporous filter membrane 247;
4) controlling the centrifuge 3 to rotate in the forward direction, wherein the rotating speed R satisfies the following conditions: working for 1min when R is 1500R/min, so that the lysate in the lysate passes through the microfiltration membrane 247 and enters the centrifugal column 26 below, and solid impurities are intercepted by the prefilter membrane 248 and the microfiltration membrane 247; due to the increase of the rotating speed, the lysate can pass through the micropore array on the micropore filter membrane 247;
5) the centrifuge 3 keeps the rotating speed R satisfying: r is 2800R/min, the operation is carried out for 3min, the first heating plate 401 is controlled to operate by the temperature control module, the paraffin valve 251 on the binding solution pre-sealing bag 254 is heated, the heating temperature is 65 ℃, the heating time is 8s, so that the paraffin valve 251 on the binding solution pre-sealing bag 254 is opened, the internal binding solution flows into the centrifugal column 26 below to be mixed with the lysate, and the nucleic acid is adsorbed by the nucleic acid adsorption membrane 261 in the centrifugal column 26; in this process, since the size of the micro-pore array on the nucleic acid adsorption film 261 is set, the binding solution, the sample, and the like on the nucleic acid adsorption film 261 do not substantially pass through the nucleic acid adsorption film 261 at this rotation speed, thereby providing space and time for mixing the binding solution and the sample, and nucleic acid is adsorbed by the nucleic acid adsorption film 261 under the action of the binding solution;
6) controlling the centrifugal machine 3 to rotate in the forward direction, wherein the rotating speed R is 5300R/min, working for 30s, then controlling the second heating sheet 402 to work through the temperature control module, heating the paraffin valve 251 on the rinsing liquid pre-sealing bag 255 at 65 ℃ for 8s, so that the paraffin valve 251 on the rinsing liquid pre-sealing bag 255 is opened, allowing the rinsing liquid inside to flow out, controlling the centrifugal machine 3 to continuously work for 3min at the current rotating speed, allowing the rinsing liquid to flow out to rinse the centrifugal column 26, and discharging the rinsing liquid from the conical outlet 262 below the centrifugal column 26; at this rotation speed, the liquid can pass through the nucleic acid adsorbing film 261, so that the rinsing liquid and the washed impurities pass through the nucleic acid adsorbing film 261 into the waste liquid collecting pipe 271;
in the processes of steps 1) -6), the sealing block 52 is always positioned above the first passage opening 502 and completely covers the first passage opening 502, the first passage opening 502 is closed, the second passage opening 503 is opened, so that the first outlet 506 is always closed, the second outlet 507 is opened, and all the waste liquid discharged from the bottom of the centrifuge tube 23 enters the waste liquid collecting tube 271 for collection;
7) controlling the reverse rotation of the centrifuge 3, and the rotating speed R of the centrifuge 3 satisfies: R9000R/min, working for 4min, so that the sealing block 52 moves above the second passage opening 503 and completely covers the second passage opening 503, the first passage opening 502 is open, and the second passage opening 503 is closed; meanwhile, the third heating sheet 403 is controlled to work by the temperature control module, the paraffin valve 251 on the eluent pre-sealing bag 256 is heated, the heating temperature is 65 ℃, the heating time is 8s, the paraffin valve 251 on the eluent pre-sealing bag 256 is opened, the internal eluent flows out to elute the centrifugal column 26, nucleic acid in the centrifugal column 26 is separated from the nucleic acid adsorption film 261, and eluted nucleic acid and the eluent flow into the nucleic acid sample collection pipe 270 together to be collected, so that a nucleic acid extracting solution is obtained, and nucleic acid extraction is completed;
8) controlling the centrifuge 3 to stop working and return to the original point, wherein all the nucleic acid sample collection tubes 270 are located right above the detection through holes 314 at the corresponding positions, and the two nucleic acid sample collection tubes 270 on the first bracket 20 are respectively located right above the two optical detection devices 6;
9) controlling the heating sleeve 41 to work through the temperature control module, heating the nucleic acid sample collecting pipe 270, and carrying out constant-temperature amplification reaction, wherein the heating temperature is 63 ℃ and the heating time is 45 min; then heating to 100 ℃, treating for 2min, carrying out enzyme inactivation treatment, and terminating the reaction;
10) after the reaction is finished, the two optical detection devices 6 are controlled to work, the two nucleic acid sample collecting pipes 270 in the same centrifugal container pipe 21 right above the detection through hole 314 at the detection station are simultaneously subjected to fluorescence detection through the two optical detection devices 6, and then the centrifuge 3 is controlled to rotate in a stepping mode, so that all the centrifugal container pipes 21 pass through the detection station one by one, the fluorescence detection of all the nucleic acid sample collecting pipes 270 is finished, and the display is carried out through the touch display screen 71.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. The utility model provides a full-automatic nucleic acid extraction, constant temperature amplification detect all-in-one which characterized in that includes:
the nucleic acid extraction and amplification integrated device comprises a nucleic acid extraction and amplification container, a centrifuge for realizing centrifugal rotation of the nucleic acid extraction and amplification container and a heating module for providing a heating function for the nucleic acid extraction and amplification container;
the optical detection module is used for carrying out fluorescence detection on a product obtained after nucleic acid extraction and isothermal amplification are completed in the nucleic acid extraction and amplification container;
the nucleic acid extraction and amplification container comprises a support which is rotatably arranged on a centrifugal station of the centrifuge and two centrifugal container tubes which are symmetrically arranged on the support, wherein each centrifugal container tube comprises an outer sleeve arranged on the support, a centrifuge tube inserted in the outer sleeve, a cracking tube inserted in the centrifuge tube, a plurality of pre-sealing liquid bags arranged on the inner wall of the centrifuge tube and positioned below the cracking tube, a centrifugal column arranged in the centrifuge tube and positioned below the pre-sealing liquid bags, and at least one collecting tube detachably connected to the bottom of the centrifuge tube;
the bottom of the pre-sealed liquid bag is provided with a liquid outlet, the liquid outlet is sealed through a paraffin valve, and the paraffin valve can be opened under the heating of the heating module, so that the liquid in the pre-sealed liquid bag flows out downwards through the liquid outlet.
2. The all-in-one machine for fully automatic nucleic acid extraction and isothermal amplification and detection according to claim 1, wherein a heat-conducting metal ring is arranged in the liquid outlet, and paraffin is filled in the heat-conducting metal ring to form the paraffin valve; a heat conducting piece in contact with the outer wall of the heat conducting metal ring is arranged inside the inner wall of the centrifugal tube;
the heating module comprises a temperature control module and a plurality of heating sheets connected with the temperature control module, the heating sheets are arranged on the inner wall of the outer sleeve and are in contact with the heat conducting piece to heat the heat conducting metal ring, so that paraffin in the heat conducting metal ring is melted, and the paraffin valve is opened;
the plurality of pre-sealing bags comprise at least one binding solution pre-sealing bag, at least one rinsing solution pre-sealing bag and at least one eluent pre-sealing bag.
3. The all-in-one machine for full-automatic nucleic acid extraction and isothermal amplification detection according to claim 2, wherein a mounting groove for mounting the heat conducting member is formed in a side wall of the centrifuge tube, the heat conducting member comprises a heat conducting column in contact with an outer wall of the heat conducting metal ring and a heat conducting sheet connected to the heat conducting column, a tail end of the heat conducting sheet is bent upwards to form an arc-shaped convex elastic sheet part, and the elastic sheet part extends out of the mounting groove and has a gap with the heat conducting sheet;
the outer wall of the heating sheet is provided with an arc-shaped groove which is used for being in matched contact with the elastic sheet part;
the heating sheets comprise a first heating sheet for heating a paraffin valve on the binding liquid pre-sealing bag, a second heating sheet for heating a paraffin valve on the rinsing liquid pre-sealing bag and a third heating sheet for heating a paraffin valve on the eluent pre-sealing bag.
4. The all-in-one machine for fully automatic nucleic acid extraction and isothermal amplification detection according to claim 3, wherein a self-switching channel block is connected to the bottom of the centrifuge tube, a self-switching channel communicated with the space below the centrifuge column is arranged in the self-switching channel block, and the self-switching channel has a first outlet and a second outlet which can be switched on/off;
the collecting pipe comprises a nucleic acid sample collecting pipe connected to the first outlet and a waste liquid collecting pipe detachably connected to the second outlet, and a PCR reaction system solution for isothermal amplification is pre-filled in the nucleic acid sample collecting pipe;
the heating module further comprises a heating sleeve connected with the temperature control module, the heating sleeve is connected to the inner wall of the bottom of the centrifuge tube and surrounds the periphery of the nucleic acid sample collecting tube, and the heating sleeve is used for heating the nucleic acid sample collecting tube;
the middle of the support is connected with a hollow rotating shaft, the hollow rotating shaft is rotatably connected to a centrifugal station of the centrifuge, a first wire hole is formed in the hollow rotating shaft, a second wire hole communicated with the first wire hole is formed in the support, a third wire hole communicated with the second wire hole is formed in the outer sleeve, and the third wire hole is communicated with the side portion of the heating piece and the side portion of the heating sleeve.
5. The full-automatic integrated machine for nucleic acid extraction and isothermal amplification and detection according to claim 4, wherein a limiting edge is arranged on the periphery of the centrifugal tube, a positioning block is connected to one side of the limiting edge close to the hollow rotating shaft, and a positioning column is connected to the bottom of the positioning block;
the surface of the bracket is provided with a positioning hole for inserting the positioning column, a touch switch is further arranged in the positioning hole, and after the positioning column is inserted into the positioning hole, the touch switch is switched from a disconnected state to a closed state, so that the electric connection among the temperature control module, the heating sheet and the heating sleeve is switched from a disconnected state to a connected state;
the bottom of the positioning hole is also provided with two pin holes communicated with the second wire guide hole, and the touch switch comprises a first pin and a second pin which are respectively inserted into the two pin holes, a connecting touch sheet which is rotatably connected with the upper end of the first pin, and a touch spring which is connected between the bottom surface of the connecting touch sheet and the bottom surface of the positioning hole;
when no external force acts on the connecting contact piece, the elastic force of the touch spring enables the connecting contact piece not to be in contact with the second pin, and the touch switch is in a disconnected state; when the positioning column is inserted into the positioning hole, the positioning column presses the connecting contact piece downwards, so that the connecting contact piece is contacted with the second pin, and the touch switch is switched to be in a conducting state.
6. The full-automatic integrated machine for nucleic acid extraction and isothermal amplification and detection according to claim 5, wherein a sealing cover is rotatably connected to a tube opening of the cracking tube, a hanging plate is connected to an inner wall of the cracking tube, a cracking solution pre-sealing bag is connected to a bottom surface of the hanging plate, at least one easy-to-tear piece is arranged on the cracking solution pre-sealing bag, a pull rope is connected to the easy-to-tear piece, and the tail end of the pull rope is connected to the bottom of the sealing cover; when the sealing cover is completely opened, the pull rope pulls the easy-to-tear piece open to break the lysate pre-sealed bag and enable the lysate to flow out of the lysate inside;
a bottom outlet of the cracking tube is connected with a porous plate, a microporous filter membrane is arranged at the upper part of the porous plate, and a pre-filter membrane is arranged above the microporous filter membrane;
the upper part of the centrifugal column is provided with a liquid distribution plate, and a plurality of liquid guide holes are densely formed in the liquid distribution plate; a nucleic acid adsorption membrane is arranged in the centrifugal column, and a conical outlet is formed in the bottom of the centrifugal column.
7. The all-in-one machine for fully automatic nucleic acid extraction and isothermal amplification and detection according to claim 6, wherein the self-switching channel comprises a switching cavity located in the middle, a plurality of liquid inlet holes communicating the switching cavity with the space below the spin column, a first liquid discharge channel communicating the switching cavity with the first outlet, and a second liquid discharge channel communicating the switching cavity with the second outlet;
a first channel port is formed at the joint of the first liquid discharge channel and the switching cavity, and a second channel port is formed at the joint of the second liquid discharge channel and the switching cavity;
the first liquid discharge channel and the second liquid discharge channel are obliquely arranged, so that the first channel opening is closer to the central axis of the centrifugal tube than the first outlet, and the second channel opening is closer to the central axis of the centrifugal tube than the second outlet;
a first spring is arranged in the switching cavity along the X direction, one end of the first spring is connected with the inner wall of the switching cavity, the other end of the first spring is connected with a sealing block which can slide in the switching cavity along the X direction, and the sealing block is used for sealing the first passage opening or the second passage opening;
when the centrifugal tube is kept still or rotates towards a first direction or a second direction but the rotating speed is less than Rt, the sealing block is positioned above the first passage port and completely covers the first passage port, the first passage port is closed, and the second passage port is opened;
when the centrifugal tube rotates towards a second direction and the rotating speed is not less than Rt, the first spring is stretched, the sealing block is positioned above the second passage opening and completely covers the second passage opening, the first passage opening is opened, and the second passage opening is closed;
wherein Rt is a rotating speed threshold value.
8. The all-in-one machine for fully automatic nucleic acid extraction and isothermal amplification detection according to claim 7, wherein the centrifuge comprises a frame, a motor disposed on the frame, a centrifugal rotating shaft drivingly connected to an output shaft of the motor, and a centrifugal turntable connected to the centrifugal rotating shaft, wherein a plurality of bracket slots for disposing the brackets are disposed on the centrifugal turntable at regular intervals, and the bracket slots form a centrifugal station of the centrifuge;
the rack comprises a base, a first supporting disc arranged on the base and a second supporting disc arranged on the first supporting disc, wherein a plurality of detection through hole groups are arranged on the second supporting disc, and each detection through hole group comprises two detection through holes;
the optical detection module comprises two optical detection devices, and two detection heads of the two optical detection devices are respectively positioned right below two detection through holes in the same detection through hole group on the detection station;
the number and the position of the detection through hole groups are matched with those of the centrifugal container tubes, so that all the centrifugal container tubes can be subjected to optical detection successively through the two optical detection modules.
9. The all-in-one machine for automatically extracting nucleic acid and amplifying and detecting nucleic acid at constant temperature according to claim 8, further comprising a housing, a cover plate arranged on the housing, a touch display screen arranged on the cover plate, and a controller arranged on the centrifugal turntable and electrically connected with the touch display screen, wherein a centrifugal machine control module and the temperature control module are embedded in the controller.
10. The integrated machine for full-automatic nucleic acid extraction and isothermal amplification detection according to claim 9, wherein the method for nucleic acid extraction and isothermal amplification detection comprises the following steps:
1) completely opening the sealing cover, pulling open the easy-to-tear piece on the lysate pre-sealing bag through the pull rope, so that the lysate in the lysate pre-sealing bag flows out and enters a cracking tube, and simultaneously adding a certain volume of sample into the cracking tube to cover the sealing cover;
2) inserting the centrifugal container tube into a bracket on a centrifugal station of a centrifugal machine, and aligning and inserting a positioning column on the centrifugal tube into a positioning hole of the bracket when the centrifugal container tube is inserted; completing the installation of all the centrifugal container pipes on a centrifugal station;
3) controlling the centrifuge to rotate in a positive and negative alternative way and to rotate at a speed R less than R0Working in a manner to perform sample lysis;
4) controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R meets the following requirements: r0≤R＜R1So that the lysate in the lysate passes through the microfiltration membrane and enters a centrifugal column below the microfiltration membrane, and solid impurities are intercepted by the pre-filtration membrane and the microfiltration membrane;
5) the centrifuge keeps the rotating speed R to meet the following conditions: r0≤R＜R1Rotating, controlling the first heating sheet to work through the temperature control module, heating a paraffin valve on the binding solution pre-sealing bag, opening the paraffin valve on the binding solution pre-sealing bag, allowing the inside binding solution to flow into a centrifugal column below to be mixed with lysate, and adsorbing nucleic acid through a nucleic acid adsorption membrane in the centrifugal column;
6) controlling the centrifugal machine to rotate in the forward direction, wherein the rotating speed R meets the following requirements: r1R is more than or equal to R and is less than Rt, the second heating sheet is controlled to work through the temperature control module, a paraffin valve on the rinsing liquid pre-sealing bag is heated, the paraffin valve on the rinsing liquid pre-sealing bag is opened, the rinsing liquid in the bag flows out to rinse the centrifugal column, and then the rinsing liquid is discharged from a conical outlet below the centrifugal column;
in the processes of steps 1) -6), the sealing block is always positioned above the first channel opening and completely covers the first channel opening, the first channel opening is closed, the second channel opening is opened, so that the first outlet is always closed, the second outlet is opened, and all the waste liquid discharged from the bottom of the centrifuge tube enters the waste liquid collecting tube to be collected;
7) controlling the reverse rotation of the centrifuge, wherein the rotating speed R of the centrifuge meets the following requirements: r is not less than R2So that the sealing block moves to the upper part of the second passage opening and completely covers the second passage openingThe first passage opening is opened, and the second passage opening is closed; controlling the third heating sheet to work through the temperature control module, heating a paraffin valve on the eluent pre-sealing bag, opening the paraffin valve on the eluent pre-sealing bag, enabling the internal eluent to flow out to elute a centrifugal column, so that nucleic acid in the centrifugal column is separated from the nucleic acid adsorption membrane, and enabling the eluted nucleic acid and the eluent to flow into the nucleic acid sample collection pipe together for collection to obtain a nucleic acid extracting solution;
wherein R is0＜R1＜Rt＜R2；
8) Controlling the centrifuge to stop working and return to the original point, wherein all the nucleic acid sample collecting pipes are positioned right above the detection through holes at the corresponding positions;
9) the heating sleeve is controlled to work by the temperature control module, the nucleic acid sample collecting pipe is heated, and constant-temperature amplification reaction is carried out;
10) after the reaction is finished, controlling the two optical detection devices to work, simultaneously carrying out fluorescence detection on two nucleic acid sample collecting pipes in the same centrifugal container pipe right above the detection through hole at the detection station through the two optical detection devices, and then controlling the centrifugal machine to rotate in a stepping manner, so that all the centrifugal container pipes pass through the detection station one by one, and completing the fluorescence detection of all the nucleic acid sample collecting pipes.
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Application publication date: 20220531 |