CN112226361B - Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method - Google Patents
Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method Download PDFInfo
- Publication number
- CN112226361B CN112226361B CN202011107595.2A CN202011107595A CN112226361B CN 112226361 B CN112226361 B CN 112226361B CN 202011107595 A CN202011107595 A CN 202011107595A CN 112226361 B CN112226361 B CN 112226361B
- Authority
- CN
- China
- Prior art keywords
- liquid
- tube
- pipe
- pcr
- rotary valve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000007788 liquid Substances 0.000 title claims abstract description 262
- 238000001514 detection method Methods 0.000 title claims abstract description 101
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 71
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 71
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 71
- 238000012546 transfer Methods 0.000 title claims abstract description 37
- 239000011324 bead Substances 0.000 title claims abstract description 36
- 238000002156 mixing Methods 0.000 claims abstract description 79
- 238000000605 extraction Methods 0.000 claims abstract description 28
- 238000010828 elution Methods 0.000 claims abstract description 26
- 230000003321 amplification Effects 0.000 claims abstract description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 23
- 238000012408 PCR amplification Methods 0.000 claims abstract description 17
- 230000009471 action Effects 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 33
- 239000012188 paraffin wax Substances 0.000 claims description 33
- 239000007787 solid Substances 0.000 claims description 31
- 238000011068 loading method Methods 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 18
- 238000005336 cracking Methods 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000009434 installation Methods 0.000 claims description 7
- 238000005382 thermal cycling Methods 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 230000002195 synergetic effect Effects 0.000 claims description 4
- 230000000903 blocking effect Effects 0.000 claims description 3
- 238000002844 melting Methods 0.000 claims description 3
- 230000008018 melting Effects 0.000 claims description 3
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 239000000523 sample Substances 0.000 description 16
- 238000005406 washing Methods 0.000 description 9
- 238000007789 sealing Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 208000035473 Communicable disease Diseases 0.000 description 4
- 239000000443 aerosol Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 210000003800 pharynx Anatomy 0.000 description 3
- 239000002985 plastic film Substances 0.000 description 3
- 229920006255 plastic film Polymers 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000002122 magnetic nanoparticle Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and a detection method, wherein the card box body is divided into a nucleic acid extraction area and an amplification detection area, the amplification detection area is provided with a rotary valve pipe, a PCR pipe, an air pump interface, an air channel and a liquid channel, the air channel respectively extends from the rotary valve pipe to a liquid mixing pipe and the air pump interface, the liquid channel respectively extends from the rotary valve pipe to an elution pipe, a liquid mixing pipe and the PCR pipe, and a rotary valve for controlling the opening and closing of the air channel and the liquid channel is arranged in the rotary valve pipe. The invention can directly add untreated clinical collection throat swabs, realizes nucleic acid extraction by externally driving the magnetic rod and the magnetic rod sleeve, realizes the switching of related liquid passages and gas passages by controlling the rotary valve, can realize the directional transfer of liquid under the action of an external pump, finally carries out real-time fluorescence PCR amplification detection in a PCR tube, and simply and efficiently realizes 'sample input and result output'.
Description
Technical Field
The invention relates to a detection device and a detection method, in particular to a nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and a detection method.
Background
Owing to the advantages of high sensitivity and high specificity of nucleic acid detection, it is increasingly used in various fields of biomedicine, in particular in the field of infectious disease detection. In the infectious disease prevention and control, the suspected case can be accurately diagnosed in time, so that precious time can be won for the epidemic prevention and control. However, nucleic acid detection requires complicated nucleic acid extraction and time-consuming PCR amplification detection steps, and requires professional experimenters to complete the detection on different experimental devices, and the whole process takes several hours, which not only greatly reduces the efficiency of nucleic acid detection, but also may cause cross contamination due to transfer of samples among different devices in the detection process, thereby affecting the detection result.
Based on above problem, some card box formula integration nucleic acid detecting systems have been released in the market at present, can draw nucleic acid and amplify the detection integration and accomplish in a card box or micro-fluidic chip, but most card boxes all adopt the theory of operation of drive liquid at present, and the card box structure is complicated, and the cost is expensive, and work efficiency is not high, to some special samples like pharynx swab sample, need loaded down with trivial details sample deactivation in earlier stage, steps such as pathogen release, can't directly add the sample and detect, have certain limitation in infectious disease detection field.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the existing liquid-driven cartridge or microfluidic chip, and provides a nucleic acid detection cartridge based on magnetic bead transfer and valve-controlled liquid transfer, which does not need manual sample processing in the early stage and really realizes full-automatic rapid nucleic acid detection of sample input and result output; another object of the present invention is to provide a method for detecting a nucleic acid detection cartridge based on magnetic bead transfer and valve-controlled pipetting.
The technical scheme is as follows: the invention provides a nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer, which comprises a box body, wherein the box body comprises a nucleic acid extraction area and an amplification detection area, and the nucleic acid extraction area comprises an elution tube; the amplification detection area is provided with a rotary valve tube, a PCR tube, an air pump interface, an air channel and a liquid channel, the air channel extends from the rotary valve tube to the liquid mixing tube and the air pump interface respectively, the liquid channel extends from the rotary valve tube to the elution tube, the liquid mixing tube and the PCR tube respectively, and a rotary valve for controlling the opening and closing of the air channel and the liquid channel is arranged in the rotary valve tube; the air pump interface is connected with an external pump, liquid is transferred to the liquid mixing tube from the elution tube under the action of the external pump to be uniformly mixed, then the liquid is transferred to the PCR tube from the liquid mixing tube, and fluorescence PCR amplification detection is carried out in the PCR tube.
The cartridge can be directly added with samples without any treatment or throat swabs collected clinically, nucleic acid extraction is realized by externally driving the magnetic rod and the magnetic rod sleeve, the switching of related liquid passages and gas passages is realized by controlling the dual-channel rotary valve, the directional transfer of liquid can be realized under the action of an external pump, and finally real-time fluorescence PCR amplification detection is carried out in a PCR tube, so that 'sample input and result output' is realized simply and efficiently. The external pump may be a plunger pump, syringe pump, air pump, etc., or other devices known in the art that can drive the transfer of liquid from the cartridge by gas pressure.
The nucleic acid extraction area comprises a self-loading magnetic rod sleeve and at least eight tube bodies, the eight tube bodies are linearly arranged and sequentially marked as No. 1-8 tubes, the self-loading magnetic rod sleeve is inserted into the No. 1 tube, the No. 2 tube is communicated with the adjacent side wall of the No. 3 tube, other tubes are mutually independent, and the No. 8 tube is an elution tube. The body is the upper end open-ended state, and the upper end flushes, and the length of No. 8 pipes is shorter, and the bottom is higher than all the other bodies, and the right side of No. 8 pipes sets up the trompil near the bottom position, connects aforementioned liquid passage. The bottom of the No. 8 tube is higher than the rest tube bodies, so that liquid can smoothly enter the PCR tube below through the liquid channel.
Heating devices are arranged below the No. 3 tube and the elution tube (No. 8 tube), and the heating devices are arranged because the steps of nucleic acid extraction cracking and elution need heating. Optionally, the PCR tube is a flat cuboid structure, and thermal cycling control devices are arranged on two sides of the PCR reaction tube. Other detection aids may be added as needed.
The kit comprises a kit body, a kit body and a kit body, wherein the kit body comprises an upper panel, a lower panel, an upper cover plate positioned on the upper surface of an amplification detection area of the upper panel, and a lower cover plate positioned on the lower surface of the amplification detection area of the lower panel; a gas channel is arranged between the upper panel and the upper cover plate, a liquid channel and a buffer pool are arranged between the lower panel and the lower cover plate, an exhaust hole penetrating through the lower panel is arranged above the buffer pool, and a PCR tube gas outlet communicated with the PCR tube is arranged below the buffer pool; when the PCR tube is filled with liquid, the gas in the tube is discharged through the gas outlet of the PCR tube, the buffer pool and the exhaust hole in sequence. Furthermore, the box body comprises a main body frame, an upper cover plate and a lower cover plate, wherein the upper cover plate is bonded on the upper surface of the amplification detection area of the upper panel in an ultrasonic, thermal, glue and other modes, and the lower cover plate is bonded on the lower surface of the amplification detection area of the lower panel in an ultrasonic, thermal, glue and other modes; the part, positioned in the amplification detection area, of the upper panel and the lower panel is cut to be half of the thickness, and channels and holes with semicircular sections are arranged on the upper surface of the upper panel and the lower surface of the lower panel and respectively form a gas channel, a liquid channel and a buffer pool together with the channels on the lower surface of the upper cover plate and the channels on the upper surface of the lower cover plate; the upper surface of the card box formed by the upper panel and the upper cover plate is provided with a pipe orifice edge bulge and a panel edge bulge, and a plastic film is installed in an ultrasonic, thermal, adhesive and other bonding modes to encapsulate liquid or solid reagents in corresponding pipe chambers.
Preferably, the self-loading magnetic rod sleeve comprises a sleeve matched with the magnetic rod, a clamping portion located above the sleeve and a stirring blade located below the sleeve, the bottom of the clamping portion is matched with the edge of the pipe orifice of the No. 1 pipe in a protruding mode, an inverted trapezoidal bayonet structure is arranged at the top of the clamping portion, and the bayonet structure is clamped with an external magnetic rod sleeve driving device. The top of the clamping part is provided with a self-loading structure, two opposite edges of the self-loading structure are lower, and the other two opposite edges of the self-loading structure are higher, so that an inverted trapezoidal bayonet structure is formed; before working, the self-loading magnetic rod sleeve is inserted into the No. 1 pipe, and the bayonet base at the top of the self-loading magnetic rod sleeve is clamped into the pipe orifice of the No. 1 pipe; during operation, the external magnetic rod sleeve driving device automatically descends and is clamped into the inverted trapezoidal bayonet structure to complete self-loading action, and then the magnetic rod sleeve can perform vertical movement and rotary movement under external driving.
Preferably, the PCR tube is provided with a liquid inlet channel, a PCR reaction tank and an exhaust channel, one end of the liquid inlet channel is communicated with the liquid channel, and the other end of the liquid inlet channel is communicated with the PCR reaction tank; one end of the exhaust channel is communicated with the air outlet hole of the PCR tube, and the other end of the exhaust channel is communicated with the PCR reaction tank; the exhaust channel is positioned above the reaction tank, and liquid enters the reaction tank from the liquid mixing pipe through the liquid inlet channel, and enters the buffer tank through the exhaust channel after being filled with the reaction tank.
Preferably, the box body is provided with a rotary valve pipe body and a liquid mixing pipe body, the upper surface of the upper cover plate is provided with an air pump interface, a rotary valve pipe orifice and a liquid mixing pipe orifice, and the lower surface of the lower cover plate is provided with a liquid mixing pipe bottom, a rotary valve pipe bottom, a PCR pipe installation position, a PCR pipe liquid inlet and a PCR pipe air outlet which are arranged at the PCR installation position; the rotary valve pipe orifice, the rotary valve pipe bottom and the rotary valve pipe body jointly form a rotary valve pipe, and the mixed liquid pipe orifice, the mixed liquid pipe bottom and the mixed liquid pipe body positioned on the main body frame jointly form a mixed liquid pipe. When the PCR tube is used, the rubber seat is arranged in the installation position of the PCR tube, and the PCR tube is inserted into the hole position of the rubber seat, so that the liquid inlet of the PCR tube is communicated with the liquid inlet channel of the PCR tube, and the gas outlet of the PCR tube is communicated with the gas exhaust channel of the PCR tube.
The pipe orifice of the rotary valve pipe extends out of the upper cover plate and is provided with a convex ring, and the convex ring is used for being matched with a concave ring on the inner side of the valve cap of the rotary valve, so that the rotary valve is limited to only do rotary motion.
Preferably, the rotary valve comprises a valve rod, three vent holes and two liquid through holes, the three vent holes are communicated with each other and form an included angle of 120 degrees in pairs, the two liquid through holes are communicated with each other and form an included angle of 120 degrees, the three liquid channels are uniformly distributed on the outer side of the rotary valve pipe around the rotary valve pipe, the included angles of the two gas channels extending to the air pump interface and the liquid mixing pipe are 120 degrees respectively, and the outlets of the vent holes and the liquid through holes on the outer surface of the valve rod correspond to the inlets of the gas channels and the liquid channels respectively. The outlet positions of the vent holes and the liquid through holes outside the valve rod meet the requirement that any two of the vent holes can be communicated with the gas channel and the liquid through holes can be communicated with two liquid channels by rotating the rotary valve when the rotary valve is inserted into the rotary valve pipe.
The two gas channels are at the same horizontal height, the three liquid channels are at the same horizontal height, and the gas channels are higher than the liquid channels; when the rotary valve is inserted into the rotary valve pipe, the position of the vent hole is at the same horizontal position with the gas channel, and the position of the liquid through hole is at the same horizontal position with the liquid channel.
Preferably, a filter element and solid paraffin are arranged in the buffer pool, and the solid paraffin is arranged above the filter element; the solid paraffin is provided with a hole for gas to pass through, and a hot cover device for softening paraffin is arranged above the exhaust hole (11D). The exhaust hole is located the top of buffer pool, and the below in exhaust hole is the buffer pool, and the below of buffer pool is PCR pipe gas outlet, and PCR pipe exhaust passage is connected to the gas outlet to exhaust when being used for flowing into liquid toward PCR pipe. The filter element is a thicker spongy body, and has the functions of ventilating and preventing the aerosol from escaping to the outside, and nucleic acid substances contained in the aerosol can pollute the environment of instruments and laboratories and influence the subsequent detection results; the paraffin is in a solid state at the beginning of the test, and the middle of the paraffin is provided with a hole, so that the smoothness of an air outlet and an exhaust hole of the PCR tube is ensured; after the sample is added, the paraffin is heated by the hot cover device above the exhaust hole, and the paraffin is covered on the surface of the buffer pool after being melted, so that the function of closing the air outlet of the PCR tube is achieved, and the follow-up PCR reaction is ensured to be carried out in a sealed environment.
The invention also provides a detection method of the nucleic acid detection card box based on magnetic bead transfer and valve-controlled liquid transfer, which comprises the following steps:
(1) before detection, reagents required by nucleic acid extraction are pre-packaged in corresponding tube bodies, PCR solid reagents are pre-packaged in a liquid mixing tube, a filter element and solid paraffin are installed in a buffer pool, and a rotary valve is adjusted to enable a liquid channel and a gas channel to be in a closed state;
(2) placing a magnetic rod sleeve in a No. 1 tube, placing a sample to be detected in a No. 2 tube, connecting an air pump interface with an external pump, starting a detection process, entering different tube chambers through the synergistic action of the external magnetic rod and the magnetic rod sleeve, realizing the transfer of magnetic beads between the No. 2 tube and the No. 8 tube, realizing the uniform mixing of the magnetic beads and a reagent through the independent up-and-down and rotating actions of the magnetic rod sleeve, completing the steps of nucleic acid cracking, combination, cleaning and elution, and finally enabling purified nucleic acid to exist in an elution tube;
(3) rotating the rotary valve, sequentially transferring the purified nucleic acid from the elution tube to the liquid mixing tube under the action of an external pump to dissolve and uniformly mix the PCR solid reagent, transferring the mixed liquid to the PCR tube, and finally performing fluorescence PCR amplification detection in the PCR tube.
The step (3) further comprises the following steps:
(a) adjusting the rotary valve to enable the liquid channel to be communicated with the elution pipe and the liquid mixing pipe, simultaneously enabling the gas channel to be communicated with the air pump interface and the liquid mixing pipe, enabling a certain amount of liquid to enter the liquid mixing pipe from the elution pipe through pumping of an external pump, and enabling the liquid to enter the liquid mixing pipe for mixing;
(b) adjusting the rotary valve to enable the liquid channel to be communicated with the liquid inlet of the liquid mixing tube and the liquid inlet of the PCR tube, and pressurizing the liquid mixing tube by an external pump to enable the PCR reaction reagent in the liquid mixing tube to enter the PCR tube and fill the reaction tank;
(c) the rotary valve is adjusted to seal the liquid inlet channel of the PCR tube, the solid paraffin in the buffer pool below is heated and melted by the hot cover device above the exhaust hole, the air outlet of the PCR tube is blocked, and the follow-up PCR reaction is ensured to be carried out in a sealed environment. Then, detection and analysis are carried out through a thermal cycling device at the bottom of the PCR tube and an optical detection device at the side surface, a detection result is obtained, and the thermal cover device continuously works in the PCR amplification process.
The hot cover device continuously works during PCR amplification, so that the paraffin is always in a liquid state, sealing is ensured, and meanwhile, the liquid in the PCR tube can be prevented from being heated and evaporated in the PCR thermal cycle process to be condensed in the buffer pool.
The specific detection method comprises the following steps:
s1, before detection starts, pre-packaging consumable materials, liquid reagents, magnetic nanoparticles, PCR solid freeze-dried reagent microspheres and the like required by nucleic acid extraction in corresponding tube chambers, thermally sealing plastic films at tube openings to prevent the liquid reagents from leaking in the transportation process, and installing a filter membrane and solid paraffin in a buffer pool, wherein the gas outlet and the exhaust hole of a PCR tube are not blocked by the paraffin; the double-channel rotary valve rotates to a corresponding angle to ensure that the liquid channel and the gas channel are both in a closed state;
the reagent type and the reagent storage position required by the detection can be adjusted according to specific detection items, and the scheme adopted by the steps is as follows: the No. 1 tube is used for placing a magnetic rod sleeve, the No. 2 and the No. 3 tube are used for storing the cracking binding liquid, the No. 4 tube is used for storing the magnetic bead liquid, the No. 5 tube is used for storing the washing liquid I, the No. 6 tube is used for storing the washing liquid II, the No. 7 tube is used for storing the alcohol removing agent, the No. 8 tube is used for storing the eluent, and the mixed liquid tube is filled with the PCR solid freeze-drying reagent;
s2, tearing a plastic sealing film of the nucleic acid extraction area, keeping the liquid mixing pipe 11B still in a sealed state, putting the pharyngeal swab which collects a patient sample into the No. 1 pipe, breaking the stem of the pharyngeal swab, loading the card box into a matched detection instrument, and connecting an external pump pipeline interface, so that full-automatic nucleic acid extraction and fluorescence PCR amplification detection can be started;
s3, the magnetic beads enter different tube chambers through the synergistic effect of an external magnetic rod and a magnetic rod sleeve, so that the magnetic beads are transferred between No. 2 tubes to No. 8 tubes, the magnetic rod sleeve independently rotates up and down to uniformly mix the magnetic beads and reagents, the steps of cracking, combining, cleaning, eluting and the like of nucleic acid are completed, and finally the purified nucleic acid exists in the No. 8 tube;
s4, rotating the double-channel rotary valve to enable the liquid channel to be communicated with the No. 8 pipe and the liquid mixing pipe, enabling the gas channel to be communicated with the external pump connector and the liquid mixing pipe, enabling a certain amount of liquid to enter the liquid mixing pipe from the No. 8 pipe through the pumping of the external pump, enabling the liquid to enter the liquid mixing pipe, dissolving the solid reagent balls, and enabling the external pump to repeatedly pump and exhaust for multiple times to promote dissolution and mixing; rotating the double-channel rotary valve to enable the liquid channel to be communicated with the liquid mixing pipe and the liquid inlet of the PCR pipe, exhausting air to the liquid mixing pipe by an external pump to enable the PCR reaction reagent in the liquid mixing pipe to enter the PCR pipe and fill the reaction tank, and then enabling the redundant liquid to enter the buffer tank through the air exhaust channel of the PCR pipe and the air outlet of the PCR pipe;
s5, rotating the double-channel rotary valve to seal the liquid inlet channel of the PCR tube, heating and melting the solid paraffin in the buffer pool below the hot cover device above the exhaust hole, and blocking the air outlet of the PCR tube to form a sealed environment in the PCR tube. Then, detection and analysis are carried out through a thermal cycling device at the bottom of the PCR tube and an optical detection device at the side surface, a detection result is obtained, and the thermal cover device is continuously heated in the PCR amplification process.
The invention principle is as follows: the invention integrates the integration of nucleic acid extraction and amplification detection, skillfully combines the magnetic rod method nucleic acid extraction based on magnetic bead transfer, liquid transfer and subsequent amplification detection through a single rotary valve, a nucleic acid sample extracted and purified by the magnetic rod method is positioned in the liquid of an elution pipe, the liquid is firstly pumped into a liquid mixing pipe and fully and uniformly mixed with a PCR solid freeze-drying reagent positioned in the liquid mixing pipe through an external air pump matched with the rotary valve, then the mixed liquid is extruded into a PCR pipe for amplification detection, and finally all steps of nucleic acid detection are fully automatically completed in a single cassette, thereby realizing the integration of nucleic acid extraction and amplification detection.
Has the advantages that: the nucleic acid detection card box based on magnetic bead transfer is suitable for automatic control, can fully automatically complete all nucleic acid detection steps including nucleic acid extraction, amplification and detection under the drive of matched instruments and equipment, and really realizes 'sample input and result output'; the invention is particularly provided with the sample adding tube, and can be directly put into samples such as throat swabs, thereby omitting fussy operations such as pathogen inactivation and release, and greatly improving the detection efficiency; the invention adopts a magnetic bead transfer-based mode to extract nucleic acid, improves the efficiency of nucleic acid extraction, simplifies the structure of the card box, and is particularly suitable for detecting infectious disease pathogens.
Drawings
FIG. 1 is an overall external view of the cartridge;
FIG. 2 is an exploded view of the cartridge as a whole;
figure 3 is an exploded view of the cartridge body;
FIG. 4 is a diagram showing the structure of the upper cover plate of the amplification detection zone;
FIG. 5 is a view showing the structure of a lower cover plate of an amplification detection zone;
FIG. 6 is a diagram of a disposable self-loading sheath;
FIG. 7 is a dual path rotary valve configuration;
FIG. 8 is a diagram showing the structure of a PCR amplification tube;
FIG. 9 is a cross-sectional view of a gas channel amplifying a detection zone;
FIG. 10 is a cross-sectional view of a liquid channel amplifying a detection region.
Wherein: 1. a cartridge body; 2. self-loading a magnetic rod sleeve; 3. a two-channel rotary valve; 4. a PCR tube; 101. a cartridge main body frame; 102. amplifying an upper cover plate of the detection area; 103. amplifying the lower cover plate of the detection area; 111. an upper panel; 112. a lower panel; 113. no. 1 tube (magnetic bar sleeve mounting tube); 114. tube No. 2 (plus sample tube); 115. tube No. 3 (cracker tube); 116. number 4 tube (magnetic bead tube); 117. tube No. 5 (wash I tube); 118. tube No. 6 (wash II tube); 119. tube No. 7 (alcohol removal tube); 11A, No. 8 tube (elution tube); 11B, a liquid mixing pipe; 11C, rotating a valve pipe; 11D, an exhaust hole; 11E, a buffer pool; 121. an air pump interface; 122. rotating the orifice of the valve pipe; 123. the pipe orifice of the liquid mixing pipe; 124. a gas channel; 131. the bottom of the liquid mixing pipe; 132. rotating the tube bottom of the valve; 133. a PCR tube mounting position; 134. a liquid channel; 135. a liquid inlet of a PCR tube; 136. a gas outlet of the PCR tube; 201. a magnetic rod sleeve; 202. a magnetic rod sleeve clamping part; 203. the magnetic rod sleeve stirring paddle; 301. rotating the valve stem; 302. rotating the bonnet; 303. rotating the raised head of the valve; 304. rotating a valve vent; 305. rotating the valve through hole; 401. a PCR tube liquid inlet channel; 402. a PCR tube exhaust channel; 403. a PCR tube reaction tank; 404. a rubber seat.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1:
as shown in FIGS. 1 to 8, the nucleic acid detecting cartridge based on magnetic bead transfer and valve-controlled liquid transfer of the present invention mainly comprises a cartridge body (cartridge body) 1, a self-loading magnetic rod sleeve 2, a dual-channel rotary valve 3 and a PCR tube 4. The card box body 1 adopts a strip-shaped layout and is provided with two strip-shaped fixed panels of an upper panel 111 and a lower panel 112 and a plurality of vertically arranged tube body structures, and the tube bodies are all provided with upper end openings. The card box body 1 can be divided into a nucleic acid extraction area and an amplification detection area according to functions, wherein the nucleic acid extraction area comprises eight tube bodies which are linearly arranged, and a square edge bulge is arranged at the tube opening of a No. 1 tube (magnetic rod sleeve installation tube) 113 and is used for limiting the direction of a self-loading magnetic rod sleeve placed in the tube opening; the adjacent side walls of the No. 2 tube (sample adding tube) 114 and the No. 3 tube (cracking tube) 115 are communicated, a sample to be detected is added from the No. 2 tube 114, is diffused to the No. 3 tube 115 after being released, and is subjected to subsequent cracking, combination and other operations under the action of the magnetic rod and the magnetic rod sleeve; the number 4-8 tubes are mutually independent, and are respectively pre-packaged with nucleic acid extraction related reagents, and the steps of washing, dealcoholizing, eluting and the like are carried out under the action of a magnetic rod and a magnetic rod sleeve.
Wherein, the No. 1 pipe 113 is provided with a magnetic rod sleeve; the tubes No. 2 and No. 3 114 and 115 store the cracking combination liquid; the No. 4 tube is a magnetic bead tube 116 and is used for storing magnetic bead liquid; the No. 5 pipe is a washing I pipe 117 and is used for storing washing liquid I; the No. 6 pipe is a washing II pipe 118 and is used for storing washing liquid II; the No. 7 pipe is an alcohol removing pipe 119 and is used for storing an alcohol removing agent; the No. 8 tube is an adsorption tube 11A and is used for storing eluent; the mixed liquid tube 11B contains a PCR solid freeze-dried reagent. Heating devices are arranged below the No. 3 tube and the elution tube (No. 8 tube), and are used for heating in the steps of cracking for nucleic acid extraction and elution. In the detection process of the nucleic acid detecting cassette of this embodiment, other auxiliary devices are all in the prior art, and are not described herein again.
As shown in fig. 6, the self-loading magnetic rod sleeve 2 comprises a sleeve 201 adapted to a magnetic rod, a clamping portion 202 located above the sleeve 201, and four stirring blades 203 located below the sleeve 201, wherein the four blades are convexly arranged on the outer wall of the sleeve; the sleeve 201 is hollow cylindrical and the magnetic rod can penetrate into the sleeve. The clamping part 202 is square, the bottom of the clamping part is matched with the projection of the square edge of the pipe orifice of the No. 1 pipe 113, a square self-loading structure is arranged at the top of the clamping part, two opposite sides are lower, the other two opposite sides are higher, so that an inverted trapezoidal bayonet structure is formed, and the bayonet structure is clamped with an external magnetic rod sleeve driving device; before working, the self-loading magnetic rod sleeve 2 is inserted into the No. 1 pipe, and the bayonet base at the top of the self-loading magnetic rod sleeve is clamped into the pipe orifice of the No. 1 pipe; during operation, the external magnetic rod sleeve driving device automatically descends and is clamped into the inverted trapezoidal bayonet structure to complete self-loading action, and then the magnetic rod sleeve can perform vertical movement and rotary movement under external driving.
As shown in FIGS. 2 and 3, the amplification detection region has a multilayer structure, and is composed of an upper plate 111, an upper cover plate 102, a lower plate 112, and a lower cover plate 103, as well as a two-channel rotary valve 3, a liquid mixing tube 11B, an air pump port 121, a PCR tube inlet port 135, and a gas channel 124 and a liquid channel 134 connecting these tube chambers. Wherein, the joint of the panels is provided with channels and holes, two gas channels 124 are formed between the upper panel 111 and the upper cover plate 102, and three liquid channels 134 and the buffer pool 11E are formed between the lower panel 112 and the lower cover plate 103. An exhaust hole 11D penetrating through the lower panel 112 is formed above the buffer tank, and a PCR tube air outlet 136 communicated with the PCR tube is formed below the buffer tank; when the PCR tube is filled with liquid, the gas in the tube is discharged through the PCR tube gas outlet 136, the buffer pool 11E and the exhaust hole 11D in sequence. The two gas channels 124 and the three liquid channels 134 all take the double-channel rotary valve 3 as the center, and the included angle between every two is 120 degrees; the gas channels in this embodiment are all at the same level, the liquid channels are also all at the same level, and the gas channels are higher than the liquid channels. The gas channel 124 is communicated with the air pump interface 121 and the liquid mixing tube 11B through the dual-channel rotary valve 3, and the liquid channel 134 is communicated with the No. 8 tube 11A, the liquid mixing tube 11B and the PCR tube liquid inlet 135 through the dual-channel rotary valve 3.
Wherein, install filter core and paraffin wax in buffer tank 11E, the filter core is in the below, and paraffin wax is above. The filter element is a thicker spongy body, and has the functions of ventilating and preventing the aerosol from escaping to the outside, and nucleic acid substances contained in the aerosol can pollute the environment of instruments and laboratories and influence the subsequent detection results; the central position of the paraffin is provided with a hole for gas to pass through. A hot cover device is arranged above the exhaust hole 11D, and automatically descends to be tightly attached to the exhaust hole when heating is needed, so that paraffin in the exhaust hole is heated and melted; the paraffin is in a solid state initially, and holes are formed in the middle of the paraffin, so that smoothness of an air outlet and an exhaust hole of the PCR tube can be guaranteed; after the sample is added, the thermal cover device above the exhaust hole 11D heats paraffin, and the paraffin is covered on the surface of the buffer pool after being melted, so that the effect of closing the air outlet of the PCR tube is achieved, and the follow-up PCR reaction is ensured to be carried out in a sealed environment.
As shown in fig. 4, the upper surface of the upper cover plate 102 is provided with an air pump interface 121 in the form of a pagoda head, a rotary valve tube nozzle 122 and a mixed liquid tube nozzle 123. As shown in FIG. 5, the lower surface of the lower cover plate 103 is provided with a mixing tube bottom 131, a rotary valve tube bottom 132, a PCR tube mounting position 133, a PCR tube liquid inlet 135 and a PCR tube gas outlet 136. The rotary valve pipe orifice 122 extends out of the upper cover plate 102 and is provided with a convex ring, the box body 1 is provided with a rotary valve pipe body and a liquid mixing pipe body, the rotary valve pipe orifice 122, the rotary valve pipe bottom 132 and the rotary valve pipe body jointly form a rotary valve pipe 11C, and the liquid mixing pipe orifice 123, the liquid mixing pipe bottom 131 and the liquid mixing pipe body positioned on the main body frame 101 jointly form a liquid mixing pipe 11B; the rubber seat 404 is arranged in the PCR tube mounting position 133, the PCR tube 4 is inserted into the hole position of the rubber seat 404, the liquid inlet of the PCR tube is communicated with the liquid inlet channel of the PCR tube, and the air outlet of the PCR tube is communicated with the air outlet channel of the PCR tube.
As shown in fig. 7, the dual channel rotary valve 3 comprises a valve stem 301, a vent hole 304, a liquid passage hole 305, a bonnet 302 and a boss 303, and accordingly, the height of the vent hole 304 is the same as the height of the gas passage, the height of the liquid passage hole 305 is the same as the height of the liquid passage, and the height of the vent hole 304 is higher than the height of the liquid passage hole. The valve rod 301 is a solid cylindrical structure, the vent hole 304 is located at a position close to the upper part of the valve rod 301, and the liquid through hole 305 is located at a position close to the bottom part of the valve rod 301. The three vent holes 304 are mutually communicated, and two vent holes form a 120-degree horizontal included angle and penetrate through the circle center of the valve pipe; the liquid through holes 305 are two in number, the insides of the liquid through holes are mutually communicated, and the liquid through holes penetrate through the circle center of the valve pipe at an included angle of 120 degrees; the vent hole 304 of the rotary valve 3 is located at the same level as the gas passage 124, and the liquid passage hole 305 is located at the same level as the liquid passage 134. The vent holes 304 and the liquid passage holes 305 are located at positions of the outlet outside the valve stem such that when the rotary valve 3 is inserted into the rotary valve tube 11C, any two of the vent holes 304 can communicate with the gas passage 124 and the liquid passage holes 305 can communicate with any two of the liquid passages 134 by rotating the rotary valve 3. Wherein, the inner wall of the valve cap 302 is provided with a circle of grooves which are matched with the convex circle of the pipe orifice 122 of the rotary valve pipe; the rotary valve projection 303 is a straight channel projection.
As shown in FIGS. 9 and 10, before the reaction, the two-channel rotary valve 3 was rotated so that the gas channel 124 and the liquid channel 134 were simultaneously closed. During reaction, firstly, the double-channel rotary valve 3 is rotated to open the gas channel 124, meanwhile, the liquid channel 134 is communicated with the elution pipe (No. 8 pipe) 11A and the liquid mixing pipe 11B, the external pump performs suction action to enable liquid to enter the liquid mixing pipe 11B from the No. 8 pipe 11A, and the external pump repeatedly performs suction and exhaust to enable the solid reagent in the liquid mixing pipe to be fully dissolved and uniformly mixed; then, the two-channel rotary valve 3 is rotated counterclockwise by 120 degrees, so that the gas channel 124 is still in an open state, meanwhile, the liquid channel is communicated with the liquid inlet 135 of the PCR tube 11B, the external pump performs air exhaust action, the extrusion liquid enters the PCR tube reaction tank 403 from the liquid mixing tube 11B through the liquid inlet 135 of the PCR tube and the liquid inlet channel 401 of the PCR tube, and the redundant liquid after filling the reaction tank 403 enters the buffer tank 11E through the air exhaust channel 402 of the PCR tube; finally, the two-way rotary valve 3 is rotated counterclockwise by 60 ° while the gas channel 124 and the liquid channel 134 are closed, and the subsequent amplification detection operation is performed. Namely, the two-channel rotary valve 3 is rotated, so that the opening and closing of a gas channel and a liquid channel can be realized; when in an open state, the rotary valve rotates 120 degrees, the gas channels are communicated, and the liquid channels can realize the communication of two adjacent liquid channels through the rotation angle.
As shown in FIG. 8, which is a structural diagram of a PCR amplification tube, the PCR reaction tube is designed to be a flat rectangular structure, and includes a liquid inlet channel 401, an air outlet channel 402 and a PCR reaction chamber 403, wherein the liquid inlet channel 401 and the air outlet channel 402 are respectively located at two sides of the PCR reaction chamber 403, and the PCR reaction chamber is located at the bottom. One end of the liquid inlet channel 401 is connected with one of the liquid channels positioned between the lower bottom plate and the lower cover plate of the card box, and the other end is connected with the PCR reaction tank; one end of the exhaust channel is connected with the exhaust hole, and the other end of the exhaust channel is connected with the PCR reaction tank. Before PCR amplification detection, liquid enters the reaction tank from the mixing pipe through the liquid inlet channel, and enters the buffer tank through the exhaust channel after filling the reaction tank. The PCR reaction tube 4 is inserted into the hole of the rubber seat, and the rubber seat 404 is arranged on the installation position of the PCR tube of the lower cover plate by heating, glue and the like. The two flat sides of the PCR reaction tube are provided with thermal cycle control devices, and the bottom surface of the PCR reaction tube can be optically detected.
Example 2:
the present embodiment provides the method for detecting nucleic acid in pharyngeal swab sample using the nucleic acid detection cartridge based on magnetic bead transfer and valve-controlled pipetting provided in embodiment 1, which comprises the following steps:
s1, before detection starts, pre-packaging consumable materials, liquid reagents, magnetic nanoparticles, PCR solid freeze-drying reagents and the like required by nucleic acid extraction in corresponding tube chambers, thermally sealing plastic films at tube openings to prevent the liquid reagents from leaking in the transportation process, and installing a filter membrane and solid paraffin in a buffer pool, wherein the air vents are not blocked by the paraffin; the double-channel rotary valve rotates to a corresponding angle to ensure that the liquid channel and the gas channel are both in a closed state;
the type of reagent and the reagent storage position required for detection can be adjusted according to specific detection items, and the scheme adopted in the embodiment is as follows: the No. 1 tube is used for placing a magnetic rod sleeve, the No. 2 and the No. 3 tube are used for storing the cracking combination liquid, the No. 4 tube is used for storing the magnetic bead liquid, the No. 5 tube is used for storing the washing liquid I, the No. 6 tube is used for storing the washing liquid II, the No. 7 tube is used for storing the alcohol removing agent, the No. 8 tube is used for storing the eluent, and the mixed liquid tube is used for storing the PCR solid freeze-drying reagent;
s2, tearing a plastic sealing film of the nucleic acid extraction area, keeping the liquid mixing pipe 11B still in a sealed state, putting the pharyngeal swab which collects a patient sample into the No. 1 pipe, breaking the stem of the pharyngeal swab, loading the card box into a matched detection instrument, and connecting an external pump pipeline interface, so that full-automatic nucleic acid extraction and fluorescence PCR amplification detection can be started;
s3, the magnetic beads enter different tube chambers through the synergistic effect of an external magnetic rod and a magnetic rod sleeve, so that the magnetic beads are transferred between No. 3 tubes to No. 8 tubes, the magnetic rod sleeve independently rotates up and down to uniformly mix the magnetic beads and a reagent, the steps of cracking, combining, cleaning, eluting and the like of nucleic acid are completed, and finally the purified nucleic acid exists in the No. 8 tube;
s4, rotating the double-channel rotary valve to enable the liquid channel to be communicated with the No. 8 pipe and the liquid mixing pipe, enabling the gas channel to be communicated with the air pump connector and the liquid mixing pipe, enabling a certain amount of liquid to enter the liquid mixing pipe from the No. 8 pipe through the suction of an external pump, enabling the liquid to enter the liquid mixing pipe, dissolving the solid reagent balls, and enabling the external pump to perform multiple suction and exhaust actions to promote dissolution and mixing; rotating the double-channel rotary valve to enable the liquid channel to be communicated with the liquid mixing pipe and the liquid inlet of the PCR pipe, exhausting air to the liquid mixing pipe by an external pump to enable PCR reaction reagents in the liquid mixing pipe to enter the PCR pipe through the liquid inlet of the PCR pipe and the liquid inlet channel of the PCR pipe and fill the reaction tank, and enabling redundant liquid to enter the buffer tank through the air exhaust channel of the PCR pipe and the air outlet of the PCR pipe;
s5, rotating the double-channel rotary valve to seal the liquid inlet channel of the PCR tube, heating and melting the solid paraffin in the buffer pool below the hot cover device above the exhaust hole, and blocking the air outlet of the PCR tube to form a sealed environment in the PCR tube. Then, detection analysis is carried out through a thermal cycling device at the bottom of the PCR tube and a side optical detection device, and a detection result is obtained.
The hot cover device continuously works during PCR amplification, so that the paraffin is always in a liquid state, sealing is ensured, and meanwhile, the liquid in the PCR tube can be prevented from being heated and evaporated in the PCR thermal cycle process to be condensed in the buffer pool.
Claims (8)
1. A nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer comprises a box body (1), and is characterized in that: the kit body comprises a nucleic acid extraction area and an amplification detection area, wherein the nucleic acid extraction area comprises an elution tube (11A), and the amplification detection area is provided with a rotary valve tube (11C), a PCR tube (4), an air pump interface (121), an air channel (124) and a liquid channel (134); the gas channel (124) extends from the rotary valve pipe (11C) to the liquid mixing pipe (11B) and the air pump interface (121), and the liquid channel (134) extends from the rotary valve pipe (11C) to the elution pipe (11A), the liquid mixing pipe (11B) and the PCR pipe (4); a rotary valve (3) for controlling the opening and closing of the gas channel and the liquid channel is arranged in the rotary valve pipe (11C); the air pump interface (121) is connected with an external pump, liquid is transferred from the elution tube (11A) to the liquid mixing tube (11B) to be uniformly mixed under the action of the external pump, then the liquid is transferred from the liquid mixing tube (11B) to the PCR tube (4), and fluorescence PCR amplification detection is carried out in the PCR tube;
the box body (1) comprises an upper panel (111), a lower panel (112), an upper cover plate (102) positioned on the upper surface of the amplification detection area of the upper panel, and a lower cover plate (103) positioned on the lower surface of the amplification detection area of the lower panel; a gas channel is arranged between the upper panel (111) and the upper cover plate (102), a liquid channel (134) and a buffer pool (11E) are arranged between the lower panel (112) and the lower cover plate (103), an exhaust hole (11D) penetrating through the lower panel (112) is arranged above the buffer pool, and a PCR tube gas outlet (136) communicated with the PCR tube is arranged below the buffer pool; when the PCR tube is filled with liquid, gas in the tube is discharged through a PCR tube gas outlet (136), a buffer pool (11E) and an exhaust hole (11D) in sequence;
the rotary valve (3) comprises a valve rod, three vent holes and two liquid through holes, wherein the three vent holes and the two liquid through holes are formed in the valve rod, the three vent holes are communicated with each other and form an included angle of 120 degrees, and the two liquid through holes are communicated with each other and form an included angle of 120 degrees; the three liquid channels (134) are uniformly distributed on the outer side of the rotary valve pipe around the rotary valve pipe, and the included angles of the two gas channels (124) which respectively extend to the air pump connector and the liquid mixing pipe are 120 degrees; the outlet of the vent hole and the liquid through hole on the outer surface of the valve rod respectively corresponds to the inlet of the gas channel and the inlet of the liquid channel.
2. The magnetic bead transfer and valve pipetting-based nucleic acid detection cartridge of claim 1, wherein: the nucleic acid extraction area comprises a self-loading magnetic rod sleeve (2) and at least eight tube bodies, wherein the eight tube bodies are linearly arranged and sequentially marked as No. 1-8 tubes, the self-loading magnetic rod sleeve (2) is inserted into the No. 1 tube (113), the No. 2 tube (114) is communicated with the adjacent side wall of the No. 3 tube (115), other tubes are mutually independent, and the No. 8 tube is an elution tube.
3. The magnetic bead transfer and valve pipetting-based nucleic acid detection cartridge of claim 2, wherein: the self-loading magnetic rod sleeve (2) comprises a sleeve matched with the magnetic rod, a clamping portion located above the sleeve and a stirring blade located below the sleeve, the bottom of the clamping portion is matched with the edge of the pipe orifice of the No. 1 pipe (113) in a protruding mode, an inverted trapezoidal bayonet structure is arranged at the top of the clamping portion, and the bayonet structure is clamped with an external magnetic rod sleeve driving device.
4. The magnetic bead transfer and valve pipetting-based nucleic acid detection cartridge of claim 1, wherein: the PCR tube (4) is provided with a liquid inlet channel (401), a PCR reaction pool (403) and an exhaust channel (402), one end of the liquid inlet channel (401) is communicated with the liquid channel (134), and the other end is communicated with the PCR reaction pool (403); one end of the exhaust channel (402) is communicated with the air outlet (136) of the PCR tube, and the other end is communicated with the PCR reaction tank (403); the exhaust channel (402) is positioned above the reaction pool, liquid enters the PCR reaction pool (403) from the liquid mixing pipe through the liquid inlet channel (401), and enters the buffer pool through the exhaust channel (402) after the reaction pool is filled.
5. The magnetic bead transfer and valve pipetting-based nucleic acid detection cartridge of claim 1, wherein: the box body (1) is provided with a rotary valve pipe body and a liquid mixing pipe body, the upper surface of the upper cover plate (102) is provided with an air pump interface (121), a rotary valve pipe orifice (122) and a liquid mixing pipe orifice (123), and the lower surface of the lower cover plate (103) is provided with a liquid mixing pipe bottom (131), a rotary valve pipe bottom (132), a PCR pipe installation position (133), a PCR pipe liquid inlet (135) and a PCR pipe gas outlet (136) which are arranged at the PCR installation position; the rotary valve pipe orifice, the rotary valve pipe bottom and the rotary valve pipe body jointly form a rotary valve pipe (11C), and the mixed liquid pipe orifice, the mixed liquid pipe bottom and the mixed liquid pipe body positioned on the main body frame jointly form a mixed liquid pipe (11B).
6. The magnetic bead transfer and valve pipetting-based nucleic acid detection cartridge of claim 1, wherein: a filter element and solid paraffin are arranged in the buffer pool, and the solid paraffin is arranged above the filter element; the solid paraffin is provided with a hole for introducing gas, and a hot cover device for softening paraffin is arranged above the exhaust hole (11D).
7. A method for detecting a nucleic acid detection cartridge based on magnetic bead transfer and valve pipetting according to claim 1, comprising the steps of:
(1) before detection, reagents required by nucleic acid extraction are pre-packaged in corresponding tube bodies, PCR solid reagents are pre-packaged in a liquid mixing tube, a filter element and solid paraffin are installed in a buffer pool, and a rotary valve is adjusted to enable a liquid channel and a gas channel to be in a closed state;
(2) placing a magnetic rod sleeve in a No. 1 tube, placing a sample to be detected in a No. 2 tube, connecting an air pump interface with an external pump, starting a detection process, entering different tube chambers through the synergistic action of the external magnetic rod and the magnetic rod sleeve, realizing the transfer of magnetic beads between the No. 2 tube and the No. 8 tube, realizing the uniform mixing of the magnetic beads and a reagent through the independent up-and-down and rotating actions of the magnetic rod sleeve, completing the steps of nucleic acid cracking, combination, cleaning and elution, and finally enabling purified nucleic acid to exist in an elution tube;
(3) rotating the rotary valve, sequentially transferring the purified nucleic acid from the elution tube to the liquid mixing tube under the action of an external pump to dissolve and uniformly mix the PCR solid reagent, transferring the mixed liquid to the PCR tube, and finally performing fluorescence PCR amplification detection in the PCR tube.
8. The method for detecting a nucleic acid detection cartridge based on magnetic bead transfer and valve pipetting of claim 7, wherein the step (3) comprises the steps of:
(a) adjusting the rotary valve to enable the liquid channel to be communicated with the elution pipe and the liquid mixing pipe, simultaneously enabling the gas channel to be communicated with the air pump interface and the liquid mixing pipe, enabling liquid to enter the liquid mixing pipe from the elution pipe through the suction of an external pump, and enabling the liquid to enter the liquid mixing pipe for mixing;
(b) adjusting the rotary valve to enable the liquid channel to be communicated with the liquid inlet of the liquid mixing tube and the liquid inlet of the PCR tube, and pressurizing the liquid mixing tube by an external pump to enable the PCR reaction reagent in the liquid mixing tube to enter the PCR tube and fill the reaction tank;
(c) adjusting the rotary valve to seal the liquid inlet channel of the PCR tube, heating and melting the solid paraffin in the buffer tank by the hot cover device, and blocking the air outlet of the PCR tube by the paraffin to form a sealed environment in the PCR tube; then, detection and analysis are carried out through a thermal cycling device at the bottom of the PCR tube and an optical detection device at the side surface, a detection result is obtained, and the thermal cover device continuously works during PCR amplification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011107595.2A CN112226361B (en) | 2020-10-15 | 2020-10-15 | Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011107595.2A CN112226361B (en) | 2020-10-15 | 2020-10-15 | Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112226361A CN112226361A (en) | 2021-01-15 |
| CN112226361B true CN112226361B (en) | 2021-11-26 |
Family
ID=74118392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011107595.2A Active CN112226361B (en) | 2020-10-15 | 2020-10-15 | Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112226361B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980650A (en) * | 2021-03-05 | 2021-06-18 | 江苏汇先医药技术有限公司 | Vertical micro-fluidic chip and method for nucleic acid extraction |
| CN112844505B (en) * | 2021-03-05 | 2024-03-08 | 江苏汇先医药技术有限公司 | Vertical microfluidic chip and method for nucleic acid extraction and amplification |
| CN113512487A (en) * | 2021-04-14 | 2021-10-19 | 新羿制造科技（北京）有限公司 | Nucleic acid extraction card box |
| CN113150945B (en) * | 2021-04-14 | 2023-01-10 | 新羿制造科技（北京）有限公司 | Slide-type nucleic acid extraction and PCR amplification detection card box |
| CN114150042B (en) * | 2022-01-07 | 2024-03-15 | 中国农业科学院农业质量标准与检测技术研究所 | Method for integrating DNA extraction and LAMP visual nucleic acid detection |
| CN114634853B (en) * | 2022-03-13 | 2023-05-02 | 首玺(广州)医疗科技有限责任公司 | Nucleic acid detection reagent device |
| CN114806802A (en) * | 2022-04-15 | 2022-07-29 | 广东润鹏生物技术有限公司 | Reagent cartridge, detection device and detection method |
| CN114570449B (en) * | 2022-04-26 | 2022-08-16 | 广州国家实验室 | Liquid transfer device and multi-path parallel liquid transfer device |
| CN114774259B (en) * | 2022-05-05 | 2023-03-21 | 东南大学 | Multichannel integrated rapid nucleic acid detection system and detection method |
| CN115141751B (en) * | 2022-09-06 | 2022-11-29 | 至美时代生物智能科技(北京)有限公司 | Nucleic acid extraction and detection device and method |
| CN115678764B (en) * | 2022-11-07 | 2023-09-08 | 苏州思迈德生物科技有限公司 | Micro-fluidic chip for rapid molecular diagnosis |
| CN117210309B (en) * | 2023-11-09 | 2024-01-26 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102947448A (en) * | 2010-06-22 | 2013-02-27 | 环球生物研究株式会社 | Composition for preventing evaporation of reaction solution during nucleic acid amplification reaction |
| CN104087508A (en) * | 2014-07-18 | 2014-10-08 | 东南大学 | Electromagnetic biological sample processing device |
| CN106573241A (en) * | 2014-06-05 | 2017-04-19 | 伊鲁米那股份有限公司 | Systems and methods including a rotary valve for at least one of sample preparation or sample analysis |
| KR20190013116A (en) * | 2017-07-31 | 2019-02-11 | 주식회사 제놀루션 | Nucleic acid extraction instrument using magnet beads and Nucleic acid extraction method using magnet beads |
| CN211170699U (en) * | 2019-11-15 | 2020-08-04 | 广州万孚生物技术股份有限公司 | Nucleic acid extraction, amplification and detection integrated device |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200032242A1 (en) * | 2018-07-30 | 2020-01-30 | Paratus Diagnostics, LLC | Multi-step processing device for nucleic acid extraction |
-
2020
- 2020-10-15 CN CN202011107595.2A patent/CN112226361B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102947448A (en) * | 2010-06-22 | 2013-02-27 | 环球生物研究株式会社 | Composition for preventing evaporation of reaction solution during nucleic acid amplification reaction |
| CN106573241A (en) * | 2014-06-05 | 2017-04-19 | 伊鲁米那股份有限公司 | Systems and methods including a rotary valve for at least one of sample preparation or sample analysis |
| CN104087508A (en) * | 2014-07-18 | 2014-10-08 | 东南大学 | Electromagnetic biological sample processing device |
| KR20190013116A (en) * | 2017-07-31 | 2019-02-11 | 주식회사 제놀루션 | Nucleic acid extraction instrument using magnet beads and Nucleic acid extraction method using magnet beads |
| CN211170699U (en) * | 2019-11-15 | 2020-08-04 | 广州万孚生物技术股份有限公司 | Nucleic acid extraction, amplification and detection integrated device |
Non-Patent Citations (2)
| Title |
|---|
| Integrated and Automated,Sample-In to Result-Out,System for Nanoechnology-Based Detection of Infectious Pathogens;Hui Chen et al.;《Nanoscience and Nanotechnology Letters》;20181031;第10卷(第10期);第1423-1428页 * |
| 基于封闭式卡盒的现场病原体检测系统的设计与实现;廖佩等;《南京医科大学学报（自然科学版）》;20170228;第37卷(第2期);第139-148页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112226361A (en) | 2021-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112226361B (en) | Nucleic acid detection card box based on magnetic bead transfer and valve control liquid transfer and detection method | |
| JP6698786B2 (en) | Single-Structure Biochip and Manufacturing Method Providing Process from Sample Introduction to Results Output | |
| US8642293B2 (en) | Disposable device for analyzing a liquid sample containing a nucleic acid with a nucleic acid amplification apparatus | |
| CN110452802A (en) | It is complete to extract molecular diagnosis micro-fluidic chip and microfluidic system | |
| US11207655B2 (en) | Microspotting device | |
| US20090227006A1 (en) | Apparatus for Performing Nucleic Acid Analysis | |
| US20100075311A1 (en) | Cartridge system | |
| CN211170699U (en) | Nucleic acid extraction, amplification and detection integrated device | |
| EP3027319B1 (en) | A fluidic cartridge and method for processing a liquid sample | |
| JP2004309145A (en) | Chemical analysis device and structure for chemical analysis | |
| JP2016028242A (en) | Fluid control processing system | |
| JP2007033350A (en) | Chemical analyzing apparatus | |
| US20170001196A1 (en) | Microfluidic based integrated sample analysis system | |
| WO2021254518A1 (en) | Sample treatment and testing apparatus | |
| CN111500675A (en) | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit | |
| CN112934285A (en) | Full-automatic totally-enclosed fluorescence quantitative PCR microfluidic diagnosis chip | |
| CN112812959A (en) | Nucleic acid extraction, amplification and detection integrated device | |
| CN112126589A (en) | Autonomous liquid suction type nucleic acid detection card box based on magnetic rod method and use method | |
| CN110777049A (en) | Microfluidic chip, nucleic acid detector and nucleic acid detection method | |
| US20220219169A1 (en) | Device and method for detecting nucleic acids in biological samples | |
| CN108728327A (en) | A kind of full-automatic sample preparation microfluidic system and preparation method thereof and application method | |
| CN212293607U (en) | One-stop full-automatic closed nucleic acid extraction and real-time fluorescence PCR test combined kit | |
| JP4593517B2 (en) | Chemical analyzer | |
| US20230151416A1 (en) | Test plate and automated biological test system | |
| CN214716737U (en) | Full-automatic totally-enclosed fluorescence quantitative PCR microfluidic diagnosis chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240322 Address after: Building 4, 4th Floor, Huale Building, No.1 Hancheng Road, Bonded Port Zone, Qingdao City, Shandong Province, 266000, and 7th Floor of Attached Buildings (B) Patentee after: Hua Xia An Jian Wu Lian Technology (Qingdao) Co.,Ltd. Country or region after: China Address before: 211102 No. 2 Southeast University Road, Jiangning District, Nanjing City, Jiangsu Province Patentee before: SOUTHEAST University Country or region before: China |
|
| TR01 | Transfer of patent right |