CN111909835A - Closed micro-fluidic nucleic acid detection card box - Google Patents
Closed micro-fluidic nucleic acid detection card box Download PDFInfo
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- CN111909835A CN111909835A CN202010863164.2A CN202010863164A CN111909835A CN 111909835 A CN111909835 A CN 111909835A CN 202010863164 A CN202010863164 A CN 202010863164A CN 111909835 A CN111909835 A CN 111909835A
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- 238000003860 storage Methods 0.000 claims abstract description 211
- 238000004140 cleaning Methods 0.000 claims abstract description 140
- 239000003480 eluent Substances 0.000 claims abstract description 136
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 94
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a closed microfluidic nucleic acid detection card box, which comprises a card box body, a cracking tube, an amplification tube, a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, a second cleaning liquid storage cavity, an eluent storage cavity, a first oil cavity, a second oil cavity, a first mechanical rotary valve, a second mechanical rotary valve, a third mechanical rotary valve, a first air source channel, a second air source channel, a third air source channel and a fourth air source channel, wherein a closed space is formed integrally. The closed card box integrating the nucleic acid extraction, amplification and detection functions integrates the two steps of nucleic acid extraction and amplification detection, is completed in a small closed box body, can be used for nucleic acid extraction and amplification detection in the environment without a molecular diagnosis laboratory, has the advantages of compact structure, easiness in operation, simple structure, easiness in assembly and the like, and effectively improves the environmental adaptability of molecular diagnosis products.
Description
Technical Field
The invention relates to a closed microfluidic nucleic acid detection card box, in particular to a closed microfluidic nucleic acid detection card box integrating nucleic acid extraction, amplification and detection functions.
Background
With the development of molecular biology, Polymerase Chain Reaction (PCR) technology for rapid in vitro amplification of a target DNA/RNA fragment is widely used and has become a main method for rapid detection of pathogen genes. The real-time fluorescence PCR technology carries out real-time monitoring on the PCR process by introducing a fluorescent dye or a specific probe marked by fluorescence, realizes the leap from qualitative judgment to quantitative analysis, can carry out high-efficiency and specific detection on pathogen genes, greatly shortens the detection window period, is favorable for realizing early diagnosis and early treatment and reduces the fatality rate.
The nucleic acid detection cycle is short, and the sensitivity and specificity are equivalent to those of the culture method, but the following problems exist: 1) the method comprises the steps of cracking clinical samples to release nucleic acid, adsorbing nucleic acid, washing impurities, eluting nucleic acid, configuring a PCR reaction system and amplifying nucleic acid, and not only has complex operation steps, but also consumes long time; 2) the combination of multiple devices requires professional laboratories and personnel, too many manual operations also easily cause cross contamination of samples, thereby causing false negative or false positive, and meanwhile, the risk of infection of inspectors is multiplied, thus seriously restricting the application and popularization of the technology.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention solves the technical problems of complicated operation steps, long time consumption, manual interference and the like of the traditional nucleic acid detection by a mode of the microfluidic card box chip.
The invention adopts the technical scheme that the closed microfluidic nucleic acid detection card box comprises a card box body, a cracking tube, an amplification tube, a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, a second cleaning liquid storage cavity, an eluent storage cavity, a first oil cavity, a second oil cavity, a first mechanical rotary valve, a second mechanical rotary valve, a third mechanical rotary valve, a first air source channel, a second air source channel, a third air source channel, a fourth air source channel, a first oil seal channel, a second oil seal channel, a third oil seal channel, a fourth oil seal channel, a first lysate transfer channel, a second lysate transfer channel, a waste liquid transfer channel, a mixing channel, a first Z-shaped reagent injection channel, a second Z-shaped reagent injection channel, a third Z-shaped reagent injection channel, a first eluent transfer channel, a second eluent transfer channel, a third eluent transfer channel, The first air source port, the second air source port and the third air source port form a closed space integrally;
further, the first Z-shaped reagent injection channel is connected with the nucleic acid extraction cavity and the first cleaning solution storage cavity, and a reagent in the first cleaning solution storage cavity can enter the nucleic acid extraction cavity along the first Z-shaped reagent injection channel under the action of an external power source; the second Z-shaped reagent injection channel is connected with the nucleic acid extraction cavity and the second cleaning solution storage cavity, and a reagent in the second cleaning solution storage cavity can enter the nucleic acid extraction cavity along the second Z-shaped reagent injection channel under the action of an external power source; the third Z-shaped reagent injection channel is connected with the nucleic acid extraction cavity and the eluent storage cavity, and a reagent in the eluent storage cavity can enter the nucleic acid extraction cavity along the third Z-shaped reagent injection channel under the action of an external power source;
furthermore, the on-off of the first air source channel, the second air source channel, the first oil seal channel, the second oil seal channel, the first lysate transfer channel and the second lysate transfer channel is switched by using a first mechanical rotary valve;
furthermore, the on-off of the first eluent transferring channel, the second eluent transferring channel, the third oil-sealed channel, the fourth oil-sealed channel, the third gas source channel and the fourth gas source channel is switched by using a second mechanical rotary valve;
further, an atmosphere port and a third air source port are designed on the third mechanical rotary valve. The third mechanical rotary valve may perform a rotary motion around the first air port. The atmospheric port is always in butt joint with the port of the nucleic acid extraction cavity to which the nucleic acid extraction cavity belongs. When the third mechanical rotary valve rotates by different angles, the third gas source port is in butt joint with a port of a first cleaning liquid storage cavity to which the first cleaning liquid storage cavity belongs, a port of a second cleaning liquid storage cavity to which the second cleaning liquid storage cavity belongs, a port of an eluent storage cavity to which the eluent storage cavity belongs, a port of a mixing cavity to which the mixing cavity belongs and a port of a waste liquid cavity to which the waste liquid cavity belongs.
Further, during nucleic acid extraction and detection, rotate first mechanical rotary valve, second mechanical rotary valve and third mechanical rotary valve to different positions, and then switch on different reagent passageways, the gas circuit passageway, oil way passageway and all kinds of cavitys, then external power supply provides power for the first gas source port of card box body upper end, second gas source port or third gas source port, and then the reagent in the control box flows, be about to transfer into nucleic acid extraction chamber by the sample of being cracked in the schizolysis pipe, accomplish the magnetic bead in nucleic acid extraction intracavity afterwards and adsorb nucleic acid, the magnetic bead washs, the waste liquid shifts to the waste liquid chamber, nucleic acid elution, shift nucleic acid to amplification pipe nucleic acid extraction and testing process, accomplish the detection in the amplification pipe at last.
The card box body is provided with a first oil cavity and a second oil cavity, sealing oil is pre-embedded in the oil cavities, and the sealing oil can be various mineral oils or similar substances which do not inhibit downstream amplification reaction and have density less than that of water; a first oil chamber port is formed in the top of the first oil chamber and communicated with the atmosphere; and a second oil chamber port is designed at the top of the second oil chamber and is communicated with the atmosphere.
The card box body is provided with a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, a second cleaning liquid storage cavity and an eluent storage cavity;
further, a nucleic acid extraction cavity port is designed at the top of the nucleic acid extraction cavity and communicated with the atmosphere;
furthermore, a waste liquid cavity port is designed at the top of the waste liquid cavity and can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
furthermore, a mixing cavity port is designed at the top of the mixing cavity and can be communicated with a second air source port to which a third mechanical rotary valve belongs;
furthermore, a first cleaning liquid storage cavity port is designed at the top of the first cleaning liquid storage cavity and can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
furthermore, a second cleaning liquid storage cavity port is designed at the top of the second cleaning liquid storage cavity and can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
furthermore, the top of the eluent storage cavity is provided with an eluent storage cavity port which can be communicated with a second gas source port belonging to a third mechanical rotary valve.
Further, the nucleic acid extraction cavity is communicated with the mixing cavity through a mixing channel;
further, the nucleic acid extraction cavity is communicated with the waste liquid cavity through a waste liquid transfer channel;
the nucleic acid extraction cavity is communicated with the first cleaning solution storage cavity through the first Z-shaped reagent injection channel;
further, the nucleic acid extraction cavity is communicated with the second cleaning solution storage cavity through a second Z-shaped reagent injection channel;
further, the nucleic acid extraction cavity is communicated with the eluent storage cavity through a third Z-shaped reagent injection channel;
the middle part of the card box body is provided with two mechanical rotary valves, namely a first mechanical rotary valve and a second mechanical rotary valve.
Furthermore, a first rotating rod installation position, a first valve inner oil seal channel, a first valve inner air source channel and a first valve inner cracking liquid transfer channel are arranged in the first mechanical rotating valve.
Furthermore, a second rotating rod installation position, an oil seal channel in the second valve, an air source channel in the second valve and an eluent transfer channel in the first valve are arranged in the second mechanical rotary valve.
When the first mechanical rotary valve is at the first position, the first air source channel and the second air source channel, the first oil seal channel and the second oil seal channel, and the first lysate transfer channel and the second lysate transfer channel are in a turn-off state;
further, when the first mechanical rotary valve is at the second position, the first oil seal channel and the second oil seal channel, and the first air source channel and the second air source channel are in an open state; the first lysate transfer channel and the second lysate transfer channel are in a shut-off state. At this time, the first oil chamber port, the first oil chamber, the first oil seal channel, the first valve inner oil seal channel, the second oil seal channel, the cracking tube, the second gas source channel, the first valve inner gas source channel, the first gas source channel and the first gas source port are communicated. At the moment, the external power source provides power to give negative pressure to the first air source port, so that the operation that sealing oil in the first oil cavity is transferred to enter the cracking tube can be realized, the cracking liquid is sealed, and aerosol pollution is prevented.
Further, when the first mechanical rotary valve is at the third position, the first gas source channel and the second gas source channel, and the first lysate transfer channel and the second lysate transfer channel are in an open state; the first oil seal passage and the second oil seal passage are still in the shut-off state. At this time, the first air source port, the first air source channel, the first valve internal air source channel, the second air source channel, the lysis tube, the first lysis tube transfer channel, the first valve internal lysis solution channel, the second lysis tube transfer channel, the nucleic acid extraction chamber port, and the third mechanical rotary valve belonging atmosphere port are communicated. At the moment, the external power source provides power to give positive pressure to the first air source port, so that the operation of transferring the lysate in the lysis tube into the nucleic acid extraction cavity can be realized.
Further, when the second mechanical rotary valve is in the first position, the first eluent transfer channel and the second eluent transfer channel, the third oil seal channel and the fourth oil seal channel, and the third air source channel and the fourth air source channel are in a shut-off state;
further, when the second mechanical rotary valve is in the second position, the first eluent transfer channel and the second eluent transfer channel, and the third gas source channel and the fourth gas source channel are in an open state; the third oil seal passage and the fourth oil seal passage are still in the shut-off state. At this time, the third gas source port, the third gas source channel, the second valve internal gas source channel, the fourth gas source channel, the amplification tube, the second eluent transfer channel, the first valve internal eluent transfer channel, the first eluent transfer channel, the nucleic acid extraction chamber port, and the third mechanical rotary valve belonging to the atmosphere port are communicated. At the moment, the external power source provides power to give negative pressure to the third gas source port, and the operation that the eluent in the nucleic acid extraction cavity is transferred into the amplification tube can be realized.
Further, when the second mechanical rotary valve is at a third position, the third oil seal channel and the fourth oil seal channel, and the third air source channel and the fourth air source channel are in an open state; the first eluent transfer channel and the second eluent transfer channel are in a shut-off state. At the moment, the third air source port, the third air source channel, the second valve inner air source channel, the fourth air source channel, the amplification pipe, the fourth oil seal channel, the second valve inner oil seal channel, the third oil seal channel, the second oil cavity and the second oil cavity port are communicated with each other. At the moment, the external power source provides power to give negative pressure to the third air source port, so that the operation that the sealing oil in the second oil cavity is transferred into the amplification tube can be realized, the amplification reagent is sealed, and aerosol pollution is prevented.
The top of the card box body is provided with a mechanical rotary valve, namely a third mechanical rotary valve.
Furthermore, a third rotating rod installation position, a second air source port and an atmosphere port are arranged in the third mechanical rotating valve.
Further, the third mechanical rotary valve performs a rotary motion around the port of the nucleic acid extraction chamber to which the nucleic acid extraction chamber belongs.
Further, when the third mechanical rotary valve is in the first position, the atmosphere port to which the third mechanical rotary valve belongs is always concentrically aligned with the port of the nucleic acid extraction chamber. In this state, the second gas source port and the waste liquid chamber port to which the waste liquid chamber belongs, the waste liquid chamber port to which the mixing chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs, and the eluent storage chamber port to which the eluent storage chamber belongs are in a shut-off state.
Further, when the third mechanical rotary valve is at the second position, the second gas source port is in a communication state with the mixing chamber port, the mixing chamber, the mixing channel, the nucleic acid extraction chamber port and the atmosphere port to which the mixing chamber belongs; at the moment, the second gas source port and the waste liquid cavity port belonging to the waste liquid cavity, the first cleaning liquid storage cavity port belonging to the first cleaning liquid storage cavity, the second cleaning liquid storage cavity port belonging to the second cleaning liquid storage cavity and the eluent storage cavity port belonging to the eluent storage cavity are in a turn-off state. At the moment, the external power source provides alternate positive pressure and negative pressure to the second air source channel, so that the mixing operation of the nucleic acid extraction cavity and the reagent in the mixing cavity can be realized.
Further, when the third mechanical rotary valve is in the third position, the second gas source port is in a communication state with the waste liquid chamber port, the waste liquid chamber, the waste liquid transfer channel, the nucleic acid extraction chamber port, and the atmosphere port to which the waste liquid chamber belongs; at the moment, the second gas source port is in a turn-off state with the port of the waste liquid cavity to which the mixing cavity belongs, the port of the first cleaning liquid storage cavity to which the first cleaning liquid storage cavity belongs, the port of the second cleaning liquid storage cavity to which the second cleaning liquid storage cavity belongs, and the port of the eluent storage cavity to which the eluent storage cavity belongs. At this time, the external power source provides negative pressure to the second air source channel, so that the transfer operation of the waste liquid in the nucleic acid extraction cavity can be realized.
Further, when the third mechanical rotary valve is at the fourth position, the second gas source port is in a communication state with the first washing liquid storage chamber port to which the first washing liquid storage chamber belongs, the first washing liquid storage chamber, the first Z-shaped reagent injection channel, the nucleic acid extraction chamber port, and the atmosphere port; at this time, the second gas source port is in a turn-off state with the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs, and the eluent storage chamber port to which the eluent storage chamber belongs. At this time, the external power source provides positive pressure to the second air source channel, so that the operation of injecting the first cleaning solution in the first cleaning solution storage cavity into the nucleic acid extraction cavity can be realized.
Further, when the third mechanical rotary valve is at the fifth position, the second gas source port is in a communication state with a second cleaning solution storage chamber port to which the second cleaning solution storage chamber belongs, the second cleaning solution storage chamber, the second Z-shaped reagent injection channel, the nucleic acid extraction chamber port, and the atmosphere port; at the moment, the second gas source port is in a turn-off state with the waste liquid cavity port to which the waste liquid cavity belongs, the mixing cavity port to which the mixing cavity belongs, the first cleaning liquid storage cavity port to which the first cleaning liquid storage cavity belongs, and the eluent storage cavity port to which the eluent storage cavity belongs. At this time, the external power source provides positive pressure to the second air source channel, so that the operation of injecting the second cleaning solution in the second cleaning solution storage cavity into the nucleic acid extraction cavity can be realized.
Further, when the third mechanical rotary valve is at the sixth position, the second gas source port is in a state of being communicated with the eluent storage cavity port, the eluent storage cavity, the third Z-shaped reagent injection channel, the nucleic acid extraction cavity port and the atmosphere port which the eluent storage cavity belongs to; at this time, the second air source port is in a turn-off state with the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, and the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs. At the moment, the external power source provides positive pressure to the second gas source channel, so that the operation of injecting the eluent in the eluent storage cavity into the nucleic acid extraction cavity can be realized.
The bottom of the card box body is provided with two trapezoidal bosses, namely a first trapezoidal boss and a second trapezoidal boss, and the two trapezoidal bosses are used for sealing and installing the cracking tube and the amplification tube.
The card box body top is provided with three air supply port, first air supply port, second air supply port, third air supply port promptly. The air source port is used as an external air source and connected into the structure of the card box, and can receive air source power generated by a peristaltic pump, an injection pump and a vacuum generator.
The second air source channel, the second oil seal channel and the first lysate transfer channel are arranged on the first trapezoid boss at the bottom of the card box body, inserted into the lysate tube and respectively used as a liquid reagent transfer channel and an air source power supply channel; the bottom of the card box body is provided with a second eluent transfer channel, a fourth oil seal channel and a fourth gas source channel on a second trapezoidal boss; inserted into the interior of the amplification tube for serving as a liquid reagent transfer and a gas source power supply channel, respectively.
The cracking tube and the amplification tube are arranged at the bottom of the card box body, and a power source is arranged at the top of the card box body and used for providing power for transferring reagents in the card box.
The cracking tube is used for high-temperature cracking of a sample, and a dry powder reagent of a cracking solution is pre-embedded in advance;
the amplification tube is used for nucleic acid adsorption and amplification detection, and amplification liquid dry powder reagents are pre-buried in advance;
the first cleaning liquid storage cavity is pre-embedded with first cleaning liquid;
and a second cleaning liquid is pre-embedded in the second cleaning liquid storage cavity.
An enclosed microfluidic nucleic acid detection cartridge can be used to separate DNA and RNA from a biological sample and perform nucleic acid amplification detection directly in an amplification tube.
Further, the nucleic acid extraction and detection step comprises:
step 1) preparation step: adding a certain volume of suspension into a clinical sample to resuspend a swab sample;
step 2) preparation step: injecting the sample obtained in the preparation step with a certain volume into a cracking tube, fully mixing the sample with a dry powder reagent of a cracking solution in the cracking tube, then installing the cracking tube on a first trapezoidal boss of a closed card box, and installing an amplification tube on a second trapezoidal boss in advance;
step 3) preparation step: the card box body is placed inside the integrated nucleic acid detector, so that the cracking tube and the amplification tube are ensured to be inserted into the heating module in an aligned mode, and correct installation of the whole card box is completed. After the card box body is placed and is accomplished, ensure first mechanical rotary valve, second mechanical rotary valve, third mechanical rotary valve and be in first position state, ensure that each reagent shifts passageway, air supply passageway, oil blanket passageway and keeps the closed state, and schizolysis pipe and amplification pipe are in the completely sealed state.
Step 4) lysis of the sample, release of nucleic acids: the driving mechanism in the integrated nucleic acid detector and the first rotating rod which the first mechanical rotating valve belongs to are arranged at the position, the first mechanical rotating valve is controlled to rotate to the second position, the external power source provides power to give negative pressure to the first air source port, the operation that the sealing oil in the first oil cavity is transferred to enter the cracking tube can be realized, a sample to be cracked in the cracking tube is sealed, and aerosol pollution in the cracking process is prevented. Then, the heating process is started, so that the sample in the lysis tube is sufficiently lysed and as much nucleic acid as possible is released.
Step 5), transferring the lysate into a nucleic acid extraction cavity: the driving mechanism in the integrated nucleic acid detector and the first rotating rod which the first mechanical rotating valve belongs to are arranged at the position, the first mechanical rotating valve is controlled to rotate to the third position, an external power source provides power to give positive pressure to the first air source port, and then the operation that lysate in the lysis tube is transferred to enter the nucleic acid extraction 8 can be realized.
Step 6) reagent mixing, nucleic acid adsorption: a driving mechanism in the integrated nucleic acid detector is connected with a third rotating rod which a third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to a second position, an external power source provides alternate positive pressure and negative pressure for a second air source channel, the mixing operation of a nucleic acid extraction cavity and a reagent in a mixing cavity can be realized, and magnetic bead dry powder pre-buried in the nucleic acid extraction cavity is fully mixed with lysis solution so as to be convenient for fully adsorbing nucleic acid;
step 7), a waste liquid transferring step I: first, a magnet is placed at the bottom of the nucleic acid extraction chamber. And then, a driving mechanism in the integrated nucleic acid detector is connected with a third rotating rod which the third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to a third position, and an external power source provides negative pressure to a second air source channel, so that the waste liquid in the nucleic acid extraction cavity can be transferred into the waste liquid cavity.
Step 8) magnetic bead washing step a: the magnet is first removed. The driving mechanism in the integrated nucleic acid detector is connected with the third rotating rod which the third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to the fourth position, and the external power source provides positive pressure to the second air source channel, so that the operation that the first cleaning solution in the first cleaning solution storage cavity is injected into the nucleic acid extraction cavity can be realized. Subsequently, step 6) is repeated to allow the washing solution to sufficiently wash the magnetic beads.
Step 9) waste liquid transfer step two: repeat step 7).
Step 10) magnetic bead washing step B: the magnet is first removed. The driving mechanism in the integrated nucleic acid detector is connected with the third rotating rod which the third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to the fifth position, the external power source provides positive pressure to the second air source channel, and the operation that the second cleaning solution in the second cleaning solution storage cavity is injected into the nucleic acid extraction cavity is realized. Subsequently, step 6) is repeated to allow the washing solution to sufficiently wash the magnetic beads.
Step 11) waste liquid transfer step three: repeat step 7).
Step 12) nucleic acid elution step: the magnet is first removed. The driving mechanism in the integrated nucleic acid detector is connected with the third rotating rod which the third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to the sixth position, and the external power source provides positive pressure to the second air source channel, so that the operation of injecting the eluent in the eluent storage cavity into the nucleic acid extraction cavity can be realized. Subsequently, step 6) is repeated to make the eluent fully contact with the magnetic beads and uniformly mix with the magnetic beads.
Step 13) transfer of the eluent: the driving mechanism in the integrated nucleic acid detector is connected with the second rotating rod which the second mechanical rotating valve belongs to, the second mechanical rotating valve is controlled to rotate to the second position, and the external power source provides power to supply negative pressure to the third gas source port, so that the operation that the eluent in the nucleic acid extraction cavity is transferred into the amplification tube can be realized. After entering the amplification tube, the nucleic acid eluent is fully mixed with the amplification solution dry powder reagent in the amplification tube to prepare for downstream nucleic acid amplification reaction.
Step 14) preparation of amplification reaction: a driving mechanism in the integrated nucleic acid detector is connected with a second rotating rod which the second mechanical rotating valve belongs to, the second mechanical rotating valve is controlled to rotate to a third position, an external power source provides power to supply negative pressure to a third air source port, the operation that sealing oil in the second oil cavity is transferred to enter the amplification tube is realized, amplification reagents are sealed, and aerosol pollution is prevented.
Step 15) amplification detection: the driving mechanism in the integrated nucleic acid detector is connected with the second rotating rod which the second mechanical rotating valve belongs to, and the second mechanical rotating valve is controlled to rotate to the first position, so that the amplification tube is isolated from the outside, and aerosol pollution is prevented.
Compared with the prior art, the invention has the beneficial effects that:
the closed card box integrating the nucleic acid extraction, amplification and detection functions integrates the two steps of nucleic acid extraction and amplification detection, is completed in a small closed box body, can be used for nucleic acid extraction and amplification detection in the environment without a molecular diagnosis laboratory, has the advantages of compact structure, easiness in operation, simple structure, easiness in assembly and the like, and effectively improves the environmental adaptability of molecular diagnosis products.
Drawings
FIG. 1 is a schematic view of a first mechanical rotary valve, a second mechanical rotary valve, and a third mechanical rotary valve of a cartridge structure of the present invention in a first position;
FIG. 2 is a schematic view of a first mechanical rotary valve of the cartridge structure of the present invention in a second position;
FIG. 3 is a schematic view of a first mechanical rotary valve of the cartridge structure of the present invention in a third position;
FIG. 4 is a schematic view of a third mechanical rotary valve of the cartridge structure of the present invention in a second position;
FIG. 5 is a schematic view of a third mechanical rotary valve of the cartridge structure of the present invention in a third position;
FIG. 6 is a schematic view of a third mechanical rotary valve of the cartridge structure of the present invention in a fourth position;
FIG. 7 is a schematic view of a third mechanical rotary valve of the cartridge structure of the present invention in a fifth position;
FIG. 8 is a schematic view of a third mechanical rotary valve of the cartridge structure of the present invention in a sixth position;
FIG. 9 is a schematic view of a second mechanical rotary valve of the cartridge structure of the present invention in a second position;
FIG. 10 is a schematic view of a second mechanical rotary valve of the cartridge structure of the present invention in a third position;
description of reference numerals: 1 is a card box body; 2 is a first trapezoidal boss; 3 is a first gas source channel; 31 is a second air source channel; 32 is a third air source channel; 33 is a fourth gas source channel; 4 is a cracking tube; 5 is a first lysate transfer channel; 51 is a second lysate transfer channel; 6 is a first oil seal channel; 61 is a second oil seal channel; 62 is a third oil seal channel; 63 is a fourth oil seal channel; 7 is a waste liquid cavity; 71 is a waste liquid chamber port; 72 is a waste liquid transfer channel; 8 is a nucleic acid extracting cavity; 81 is a nucleic acid extraction chamber port; 9 is a mixing cavity; 91 is a mixing chamber port; 92 is a mixing channel; 10 is a first eluent transferring channel; 101 is a second eluent transfer channel; 11 is an amplification tube; 12 is a first air supply port; 13 is a third gas supply port; 14 is a first oil chamber; 141 is a first oil chamber port; 15 is a second oil chamber; 151 is a second oil chamber port; 16 is a first mechanical rotary valve; 161 is the first rotating rod mounting place; 17 is a second mechanical rotary valve; 171 is a second rotating lever mounting place; 18 is a third mechanical rotary valve; 181 is the third rotating rod mounting position; 182 is an atmospheric port; 183 is a second gas supply port; 19 is a first valve inner oil seal channel; 20 is a first in-valve gas source passage; 21 is a first in-valve cracking liquid transfer channel; 22 is a first cleaning liquid storage chamber; 221 is a first cleaning solution storage chamber port; 222 is a first Z-shaped reagent injection channel; 23 is a second cleaning liquid storage chamber; 231 is a second cleaning liquid storage chamber port; 232 is a second Z-shaped reagent injection channel; 24 is an eluent storage cavity; 241 is an eluent storage cavity port; 242 is a third Z-type reagent injection channel; 25 is a second valve inner oil seal channel; 26 is an eluent transfer channel in the first valve; 27 is a second valve internal gas source passage; 28 is a second trapezoidal boss; and 29 is a magnet.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1
As shown in FIG. 1-2, the kit comprises a cartridge body 1, a lysis tube 4, an amplification tube 11, a nucleic acid extraction chamber 8, a waste liquid chamber 7, a mixing chamber 9, a first cleaning liquid storage chamber 22, a second cleaning liquid storage chamber 23, an eluent storage chamber 24, a first oil chamber 14, a second oil chamber 15, a first mechanical rotary valve 16, a second mechanical rotary valve 17, a third mechanical rotary valve 18, a first gas source channel 3, a second gas source channel 31, a third gas source channel 32, a fourth gas source channel 33, a first oil-sealed channel 6, a second oil-sealed channel 61, a third oil-sealed channel 62, a fourth oil-sealed channel 63, a first lysis liquid transfer channel 5, a second lysis liquid transfer channel 51, a waste liquid transfer channel 72, a mixing channel 92, a first Z-type reagent injection channel 222, a second Z-type reagent injection channel 232, a third Z-type reagent injection channel 242, a first eluent transfer channel 10, a second oil-sealed channel 18, a third oil-sealed channel 63, a, The second eluent transfer channel 101, first gas source port 12, second gas source port 183, and third gas source port 13, form an enclosed volume.
The cracking tube 4 can be used for high-temperature cracking of samples, and a lysate dry powder reagent is placed in the cracking tube in advance. The amplification tube 11 can be used for nucleic acid adsorption and amplification detection, and amplification solution dry powder reagents are placed in the amplification tube in advance. The first cleaning solution storage chamber 22 is pre-embedded with 70% ethanol. The second cleaning solution storage chamber 23 is pre-buried with deionized water. The eluent storage chamber 24 is pre-filled with eluent.
The card box body 1 is arranged in the integrated nucleic acid detector before testing, so that the cracking tube 4 and the amplification tube 11 are ensured to be aligned and inserted into the heating module, and the correct installation of the whole card box is completed. The first rotating rod mounting part 161 of the first mechanical rotary valve 16, the first rotating rod mounting part 171 of the second mechanical rotary valve 17 and the second rotating rod mounting part 181 of the third mechanical rotary valve 18 can be connected with a driving mechanism in the integrated nucleic acid detector to complete rotary motion, and are used for switching different reagent channels, gas source channels and oil seals. The top of the card box body 1 is provided with a power source which can provide required power for transferring the reagent in the card box.
By rotating the first mechanical rotary valve 16 to different positions, the first and second gas supply passages 3 and 31, the first and second oil-sealed passages 6 and 62, and the first and second lysate transfer passages 5 and 51 can be controlled to be in the off or on state.
The first 10 and second 101 eluent transfer channels, the third 62 and fourth 63 oil-sealed channels, and the third 32 and fourth 33 gas source channels can be controlled to be in either an off or on state by rotating the second mechanical rotary valve 17 to different positions.
The third mechanical rotary valve 18 rotates around the nucleic acid extraction chamber port 81 to which the nucleic acid extraction chamber 8 belongs, that is, the atmosphere port 182 to which the third mechanical rotary valve 18 belongs is always concentrically aligned with the nucleic acid extraction chamber port 81. When the third mechanical rotary valve 18 is moved to different positions, the second gas source port 183 of the third mechanical rotary valve 18 can be controlled to be in an open or closed state with the waste liquid chamber port 71 of the waste liquid chamber 7, the waste liquid chamber port 91 of the mixing chamber 9, the first cleaning liquid storage chamber port 221 of the first cleaning liquid storage chamber 22, the second cleaning liquid storage chamber port 231 of the second cleaning liquid storage chamber 23, and the eluent storage chamber port 241 of the eluent storage chamber 24, respectively.
During nucleic acid extraction and detection, rotate first mechanical rotary valve 16, second mechanical rotary valve 17 and third mechanical rotary valve 18 to different positions, and then switch on different reagent passageways, the gas circuit passageway, oilway passageway and all kinds of cavitys, then external power supply provides power for first gas source port 12, second gas source port 183 or third gas source port 13 of card box body 1 upper end, and then the reagent in the control card box body 1 flows, be about to shift into nucleic acid extraction chamber 8 by the sample of schizolysis in the schizolysis pipe 4, accomplish magnetic bead absorption nucleic acid in nucleic acid extraction chamber 8 afterwards, the magnetic bead washs, the waste liquid is shifted to the waste liquid chamber, nucleic acid elution, shift nucleic acid to nucleic acid extraction and detection processes such as amplication pipe, the detection is accomplished in amplification pipe 11 at last.
According to one embodiment of the present invention, the operation of the closed cartridge is as follows:
1) the preparation method comprises the following steps: adding a certain volume of suspension into a clinical sample to resuspend a swab sample;
2) the preparation method comprises the following steps: injecting a certain volume of sample obtained in the preparation step 1 into the cracking tube 4, fully mixing the sample with a dry powder reagent of a cracking solution in the cracking tube 4, then installing the cracking tube 4 on the first trapezoidal boss 2 of the closed card box, and installing the amplification tube 11 in advance at the second trapezoidal boss 28;
3) the preparation method comprises the following steps: the card box body 1 is placed in the integrated nucleic acid detector, so that the cracking tube 4 and the amplification tube 11 are ensured to be aligned and inserted into the heating module, and the correct installation of the whole card box is completed. The first rotating lever attachment portion 161 to which the first mechanical rotary valve 16 belongs, the first rotating lever attachment portion 171 to which the second mechanical rotary valve 17 belongs, and the third rotating lever attachment portion 181 to which the third mechanical rotary valve 18 belongs may be connected to a drive mechanism inside the integrated nucleic acid detecting apparatus. After the cartridge body 1 is placed, the first mechanical rotary valve 16, the second mechanical rotary valve 17 and the third mechanical rotary valve 18 are ensured to be in the first position state, the reagent transfer channels, the gas source channel and the oil seal channel are ensured to be kept in the closed state, and the cracking tube 4 and the amplification tube 11 are in the completely sealed state.
4) Lysis of the sample, release of nucleic acids: the driving mechanism in the integrated nucleic acid detecting instrument and the first rotating rod mounting part 161 of the first mechanical rotating valve 16 control the first mechanical rotating valve 16 to rotate to the second position, the first oil seal channel 6 and the second oil seal channel 61, and the first air source channel 3 and the second air source channel 31 are in the open state; the first lysate transfer channel 5 and the second lysate transfer channel 51 are in the off state. At this time, the first oil chamber port 141, the first oil chamber 14, the first oil seal passage 6, the first in-valve oil seal passage 19, the second oil seal passage 61, the cleavage tube 4, the second gas source passage 31, the first in-valve gas source passage 20, the first gas source passage 3, and the first gas source port 12 communicate with each other. At this time, the external power source provides power to apply negative pressure to the first air source port 12, so that the operation of transferring the sealing oil in the first oil chamber 14 into the cracking tube 4 can be realized, the sample to be cracked in the cracking tube 4 can be sealed, and aerosol pollution in the cracking process can be prevented. Then, the driving mechanism in the integrated nucleic acid detecting instrument and the first rotating rod mounting part 161 to which the first mechanical rotating valve 16 belongs control the first mechanical rotating valve 16 to rotate to the first position, so that the first air source channel 3 and the second air source channel 31, the first oil seal channel 6 and the second oil seal channel 61, and the first lysate transferring channel 5 and the second lysate transferring channel 51 are in a shut-off state, and the lysis tube 4 is completely isolated from the outside. Then, the heating process is started, so that the sample in the lysis tube 6 is sufficiently lysed and as much nucleic acid as possible is released.
5) Transferring the lysate into a nucleic acid extraction cavity: a driving mechanism in the integrated nucleic acid detector and a first rotating rod mounting part 161 of a first mechanical rotating valve 16 control the first mechanical rotating valve 16 to rotate to a third position, so that a first gas source channel 3, a second gas source channel 31, a first lysate transfer channel 5 and a second lysate transfer channel 51 are in an open state; the first oil seal passage 6 and the second oil seal passage 61 are still in the shut-off state. At this time, the first gas source port 12, the first gas source channel 3, the first valve internal gas source channel 20, the second gas source channel 31, the cleavage tube 4, the first cleavage tube transfer channel 5, the first valve internal cleavage solution channel 21, the second cleavage tube transfer channel 51, the nucleic acid extraction chamber 8, the nucleic acid extraction chamber port 81, and the atmosphere port 182 to which the third mechanical rotary valve 18 belongs are communicated. At this time, the external power source provides power to provide positive pressure to the first air source port 12, so that the operation of transferring the lysis solution in the lysis tube 4 into the nucleic acid extraction cavity 8 can be realized. Then, the driving mechanism in the integrated nucleic acid detecting instrument and the first rotating rod mounting part 161 to which the first mechanical rotating valve 16 belongs control the first mechanical rotating valve 16 to rotate to the first position, so that the first air source channel 3 and the second air source channel 31, the first oil seal channel 6 and the second oil seal channel 61, and the first lysate transferring channel 5 and the second lysate transferring channel 51 are in a shut-off state, and the lysis tube 4 is completely isolated from the outside.
6) Mixing reagents, nucleic acid adsorption: the driving mechanism in the integrated nucleic acid detecting instrument is connected with the third rotating rod mounting part 181 to which the third mechanical rotating valve 18 belongs, and controls the third mechanical rotating valve 18 to rotate to the second position, and the second gas source port 183 is in a communication state with the mixing chamber port 91 to which the mixing chamber 9 belongs, the mixing chamber 9, the mixing channel 92, the nucleic acid extracting chamber 8, the nucleic acid extracting chamber port 81, and the atmosphere port 182 to which the third mechanical rotating valve 18 belongs; at this time, the second gas source port 183 is in a shut-off state with the waste liquid chamber port 71 to which the waste liquid chamber 7 belongs, the first cleaning liquid storage chamber port 221 to which the first cleaning liquid storage chamber 22 belongs, the second cleaning liquid storage chamber port 231 to which the second cleaning liquid storage chamber 23 belongs, and the eluent storage chamber port 241 to which the eluent storage chamber 24 belongs. At this time, the external power source provides alternate positive pressure and negative pressure to the second air source channel 183, so that the mixing operation of the reagents in the nucleic acid extraction cavity 8 and the mixing cavity 9 can be realized, and the magnetic bead dry powder pre-embedded in the nucleic acid extraction cavity 8 is fully mixed with the lysate, so that the nucleic acid can be fully adsorbed;
7) waste liquid transfer step 1: first, the magnet 29 is placed at the bottom of the nucleic acid extraction chamber 8. Then, a driving mechanism in the integrated nucleic acid detecting instrument is connected with a third rotating rod mounting part 181 to which a third mechanical rotating valve 18 belongs, the third mechanical rotating valve 18 is controlled to rotate to a third position, and a second gas source port 183 is in a communication state with a waste liquid cavity port 71 to which a waste liquid cavity 7 belongs, the waste liquid cavity, a waste liquid transfer channel 7, a nucleic acid extraction cavity 8, a nucleic acid extraction cavity port 81 and an atmosphere port 182; at this time, the second gas source port 183 is in a shut-off state from the mixing chamber port 91 of the mixing chamber 9, the first cleaning liquid storage chamber port 221 of the first cleaning liquid storage chamber 22, the second cleaning liquid storage chamber port 231 of the second cleaning liquid storage chamber 23, and the eluent storage chamber port 241 of the eluent storage chamber 24. At this time, the external power source provides negative pressure to the second air source channel 183, so that the waste liquid in the nucleic acid extraction chamber 8 can be transferred into the waste liquid chamber 7.
8) Magnetic bead washing step 1: the magnet 29 is first removed. The driving mechanism in the integrated nucleic acid detecting instrument is connected to the third rotating rod mounting part 181 to which the third mechanical rotating valve 18 belongs, and controls the third mechanical rotating valve 18 to rotate to the fourth position, and the second gas source port 183 is in a state of being communicated with the first wash solution storage chamber port 221 to which the first wash solution storage chamber 22 belongs, the first wash solution storage chamber 22, the first Z-shaped reagent injection channel 222, the nucleic acid extraction chamber 8, the nucleic acid extraction chamber port 81, and the atmosphere port 182; at this time, the second gas source port 183 is in a shut-off state with the waste liquid chamber port 71 to which the waste liquid chamber 7 belongs, the mixing chamber port 91 to which the mixing chamber 9 belongs, the second cleaning liquid storage chamber port 231 to which the second cleaning liquid storage chamber 23 belongs, and the eluent storage chamber port 241 to which the eluent storage chamber 24 belongs. At this time, the external power source provides positive pressure to the second gas source channel 183, so that the operation of injecting the first washing solution in the first washing solution storage chamber 22 into the nucleic acid extraction chamber 8 can be achieved. Subsequently, the content of step 6 is repeated to make the washing liquid to sufficiently wash the magnetic beads.
9) Waste liquid transfer step 2: the contents of step 7 are repeated.
10) Magnetic bead cleaning step 2: the magnet 29 is first removed. The driving mechanism in the integrated nucleic acid detecting instrument is connected with the third rotating rod mounting part 181 to which the third mechanical rotating valve 18 belongs, and controls the third mechanical rotating valve 18 to rotate to the fifth position, and the second gas source port 183 is in a communication state with the second wash solution storage chamber port 231 to which the second wash solution storage chamber 23 belongs, the second wash solution storage chamber 23, the second Z-shaped reagent injection channel 232, the nucleic acid extraction chamber 8, the nucleic acid extraction chamber port 81, and the atmosphere port 182; at this time, the second gas source port 183 is in a shut-off state with the waste liquid chamber port 71 to which the waste liquid chamber 7 belongs, the mixing chamber port 91 to which the mixing chamber 9 belongs, the first washing liquid storage chamber port 221 to which the first washing liquid storage chamber 22 belongs, and the eluent storage chamber port 241 to which the eluent storage chamber 24 belongs. At this time, the external power source provides positive pressure to the second gas source channel 183, so that the operation of injecting the second washing solution in the second washing solution storage chamber 23 into the nucleic acid extraction chamber 8 can be achieved. Subsequently, the content of step 6 is repeated to make the washing liquid to sufficiently wash the magnetic beads.
11) Waste liquid transfer step 3: the contents of step 7 are repeated.
12) Nucleic acid elution step: the magnet 29 is first removed. A driving mechanism in the integrated nucleic acid detector is connected with a third rotating rod mounting part 181 to which the third mechanical rotating valve 18 belongs, the third mechanical rotating valve 18 is controlled to rotate to a sixth position, and the second gas source port 183 is communicated with an eluent storage cavity port 241 to which the eluent storage cavity 24 belongs, the eluent storage cavity 24, the third Z-shaped reagent injection channel 242, the nucleic acid extraction cavity 8, a nucleic acid extraction cavity port 81 and an atmosphere port 182; at this time, the second air supply port 183 is in a shut-off state with the waste liquid chamber port 71 to which the waste liquid chamber 7 belongs, the mixing chamber port 91 to which the mixing chamber 9 belongs, the first cleaning liquid storage chamber port 221 to which the first cleaning liquid storage chamber 22 belongs, and the second cleaning liquid storage chamber port 231 to which the second cleaning liquid storage chamber 23 belongs. At this time, the external power source provides positive pressure to the second gas source channel 183, so that the operation of injecting the eluent in the eluent storage chamber 24 into the nucleic acid extraction chamber 8 can be realized. Subsequently, the content of the step 6 is repeated to make the eluent fully contact with the magnetic beads and uniformly mix with the magnetic beads. Before the next operation, the driving mechanism in the integrated nucleic acid detecting instrument is connected to the third rotating rod mounting part 181 to which the third mechanical rotating valve 18 belongs, and controls the third mechanical rotating valve 18 to rotate to the first position, in this state, the second gas source port 183 is in a shut-off state with the waste liquid chamber port 71 to which the waste liquid chamber 7 belongs, the waste liquid chamber port 91 to which the mixing chamber 9 belongs, the first washing liquid storage chamber port 221 to which the first washing liquid storage chamber 22 belongs, the second washing liquid storage chamber port 231 to which the second washing liquid storage chamber 23 belongs, and the eluent storage chamber port 241 to which the eluent storage chamber 24 belongs.
13) Transferring the eluent: the driving mechanism in the integrated nucleic acid detecting instrument is connected with the second rotating rod installation position 171 where the second mechanical rotating valve 17 belongs to, and controls the second mechanical rotating valve 17 to rotate to the second position, and the first eluent transferring channel 10, the second eluent transferring channel 101, the third gas source channel 32 and the fourth gas source channel 33 are in an opening state; the third oil seal passage 62 and the fourth oil seal passage 63 are still in the shut-off state. At this time, the third gas source port 13, the third gas source channel 32, the second valve internal gas source channel 27, the fourth gas source channel 33, the amplification tube 11, the second eluent transfer channel 101, the first valve internal eluent transfer channel 26, the first eluent transfer channel 10, the nucleic acid extraction chamber 8, the nucleic acid extraction chamber port 81, and the atmosphere port 182 to which the third mechanical rotary valve 18 belongs are communicated. At this time, the external power source provides power to apply negative pressure to the third gas source port 13, so that the operation of transferring the eluent in the nucleic acid extraction cavity 8 into the amplification tube 11 can be realized. After entering the amplification tube 11, the nucleic acid eluent is fully mixed with the amplification solution dry powder reagent in the amplification tube 11 to prepare for downstream nucleic acid amplification reaction.
14) Preparation of amplification reaction: the driving mechanism in the integrated nucleic acid detecting instrument is connected with the second rotating rod mounting part 171 where the second mechanical rotating valve 17 belongs, and controls the second mechanical rotating valve 17 to rotate to the third position, and the third oil seal channel 62, the fourth oil seal channel 63, the third air source channel 32 and the fourth air source channel 33 are in an open state; the first eluent transfer channel 10 and the second eluent transfer channel 101 are in a shut-off state. At this time, the third air supply port 13, the third air supply passage 32, the second in-valve air supply passage 27, the fourth air supply passage 33, the amplification tube 11, the fourth oil seal passage 63, the second in-valve oil seal passage 25, the third oil seal passage 62, the second oil chamber 15, the second oil chamber port 151 communicate with each other. At this time, the external power source provides power to apply negative pressure to the third air source port 13, so that the operation of transferring the sealing oil in the second oil chamber 15 into the amplification tube 11 can be realized, the amplification reagent can be sealed, and aerosol pollution can be prevented.
15) And (3) amplification detection: the driving mechanism in the integrated nucleic acid detecting instrument is connected with the second rotating rod mounting part 171 where the second mechanical rotating valve 17 belongs, the second mechanical rotating valve 17 is controlled to rotate to the first position, the third oil seal channel 62, the fourth oil seal channel 63, the third air source channel 32, the fourth air source channel 33, the first eluent transferring channel 10 and the second eluent transferring channel 101 are in a cut-off state, the amplification tube 11 is ensured to be isolated from the outside, and aerosol pollution is prevented.
The closed microfluidic nucleic acid detection card box integrating the nucleic acid extraction, amplification and detection functions can be used for nucleic acid extraction and amplification detection in the environment without a molecular diagnosis laboratory, and has the advantages of compact structure, easiness in operation, high environmental tolerance, capability of detecting multiple indexes and the like.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A closed microfluidic nucleic acid detection cartridge, comprising: comprises a card box body (1), a cracking tube (4), an amplification tube (11), a nucleic acid extraction cavity (8), a waste liquid cavity (7), a mixing cavity (9), a first cleaning liquid storage cavity (22), a second cleaning liquid storage cavity (23), an eluent storage cavity (24), a first oil cavity (14), a second oil cavity (15), a first mechanical rotary valve (16), a second mechanical rotary valve (17), a third mechanical rotary valve (18), a first air source channel (3), a second air source channel (31), a third air source channel (32), a fourth air source channel (33), a first oil seal channel (6), a second oil seal channel (61), a third oil seal channel (62), a fourth oil seal channel (63), a first cracking liquid transfer channel (5), a second cracking liquid transfer channel (51), a waste liquid transfer channel (72), a mixing channel (92), a first Z-type reagent injection channel (222), A second Z-type reagent injection channel (232), a third Z-type reagent injection channel (242), a first eluent transfer channel (10), a second eluent transfer channel (101), a first gas source port (12), a second gas source port (183), and a third gas source port (13), the cartridge integrally forming an enclosed space;
the first Z-shaped reagent injection channel (222) is connected with the nucleic acid extraction cavity (8) and the first cleaning solution storage cavity (22), and a reagent in the first cleaning solution storage cavity (22) can enter the nucleic acid extraction cavity (8) along the first Z-shaped reagent injection channel (222) under the action of an external power source; the second Z-shaped reagent injection channel (232) is connected with the nucleic acid extraction cavity (8) and the second cleaning solution storage cavity (23), and the reagent in the second cleaning solution storage cavity (23) can enter the nucleic acid extraction cavity (8) along the second Z-shaped reagent injection channel (232) under the action of an external power source; the third Z-shaped reagent injection channel (242) is connected with the nucleic acid extraction cavity (8) and the eluent storage cavity (24), and the reagent in the eluent storage cavity (24) can enter the nucleic acid extraction cavity (8) along the third Z-shaped reagent injection channel (242) under the action of an external power source; the on-off of the first air source channel (3), the second air source channel (31), the first oil seal channel (6), the second oil seal channel (61), the first lysate transfer channel (5) and the second lysate transfer channel (51) are switched by using a first mechanical rotary valve (16);
the on-off of the first eluent transferring channel (10), the second eluent transferring channel (101), the third oil-sealed channel (62), the fourth oil-sealed channel (63), the third air source channel (32) and the fourth air source channel (33) are switched by using a second mechanical rotary valve (17);
the third mechanical rotary valve (18) is provided with an atmosphere port (182) and a third air source port (13), and the third mechanical rotary valve (18) can rotate around the second atmosphere port (182); the atmospheric port (182) is always in butt joint with the nucleic acid extraction cavity port (81) of the nucleic acid extraction cavity (8); when the third mechanical rotary valve (18) rotates at different angles, the third gas source port (13) is mutually butted with a first cleaning liquid storage cavity port (221) belonging to the first cleaning liquid storage cavity (22), a second cleaning liquid storage cavity port (231) belonging to the second cleaning liquid storage cavity (23), an eluent storage cavity port (241) belonging to the eluent storage cavity (24), a mixing cavity port (91) belonging to the mixing cavity (9) and a waste liquid cavity port (71) belonging to the waste liquid cavity (7) respectively;
when nucleic acid is extracted and detected, rotate first mechanical rotary valve (16), second mechanical rotary valve (17) and third mechanical rotary valve (18) to different positions, and then switch on different reagent passageway, the gas circuit passageway, oilway passageway and all kinds of cavitys, then external power supply provides power for first gas source port (12) of card box body (1) upper end, second gas source port (183) or third gas source port (13), and then the reagent in the control card box flows, be about to transfer into nucleic acid extraction chamber (8) by the schizolysis sample in schizolysis pipe (4), accomplish the magnetic bead in nucleic acid extraction chamber (8) afterwards and adsorb nucleic acid, the magnetic bead washs, the waste liquid shifts waste liquid to waste liquid chamber (7), nucleic acid elution, transfer nucleic acid to amplification pipe (11) nucleic acid extraction and testing process, at last accomplish the detection in amplification pipe (11).
2. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the card box body is provided with a first oil cavity and a second oil cavity, sealing oil is pre-embedded in the oil cavities, and the sealing oil can be various mineral oils or similar substances which do not inhibit downstream amplification reaction and have density less than that of water; a first oil chamber port is formed in the top of the first oil chamber and communicated with the atmosphere; and a second oil chamber port is designed at the top of the second oil chamber and is communicated with the atmosphere.
3. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the card box body is provided with a nucleic acid extraction cavity, a waste liquid cavity, a mixing cavity, a first cleaning liquid storage cavity, a second cleaning liquid storage cavity and an eluent storage cavity;
the top of the nucleic acid extraction cavity is provided with a nucleic acid extraction cavity port which is communicated with the atmosphere;
the top of the waste liquid cavity is provided with a waste liquid cavity port which can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
the top of the mixing cavity is provided with a mixing cavity port which can be communicated with a second air source port to which a third mechanical rotary valve belongs;
a first cleaning liquid storage cavity port is formed in the top of the first cleaning liquid storage cavity and can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
a second cleaning liquid storage cavity port is formed in the top of the second cleaning liquid storage cavity and can be communicated with a second gas source port to which a third mechanical rotary valve belongs;
and the top of the eluent storage cavity is provided with an eluent storage cavity port which can be communicated with a second gas source port to which a third mechanical rotary valve belongs.
The nucleic acid extraction cavity is communicated with the mixing cavity through the mixing channel;
the nucleic acid extraction cavity is communicated with the waste liquid cavity through a waste liquid transfer channel;
the nucleic acid extraction cavity is communicated with the first cleaning solution storage cavity through the first Z-shaped reagent injection channel;
the nucleic acid extraction cavity is communicated with the second cleaning solution storage cavity through a second Z-shaped reagent injection channel;
the nucleic acid extraction cavity is communicated with the eluent storage cavity through a third Z-shaped reagent injection channel.
4. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the middle part of the card box body is provided with two mechanical rotary valves, namely a first mechanical rotary valve and a second mechanical rotary valve;
a first rotating rod mounting position, a first valve inner oil seal channel, a first valve inner air source channel and a first valve inner cracking liquid transfer channel are arranged in the first mechanical rotating valve;
and a second rotating rod mounting part, an oil seal channel in the second valve, an air source channel in the second valve and an eluent transfer channel in the first valve are arranged in the second mechanical rotary valve.
5. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
when the first mechanical rotary valve is at the first position, the first air source channel and the second air source channel, the first oil seal channel and the second oil seal channel, and the first lysate transfer channel and the second lysate transfer channel are in a turn-off state;
when the first mechanical rotary valve is at the second position, the first oil seal channel and the second oil seal channel, and the first air source channel and the second air source channel are in a communicated state; the first lysate transfer channel and the second lysate transfer channel are in a cut-off state, at the moment, the port of the first oil cavity, the first oil seal channel, the oil seal channel in the first valve, the second oil seal channel, the cracking tube, the second gas source channel, the gas source channel in the first valve, the first gas source channel and the first gas source port are communicated, at the moment, an external power source provides power to give negative pressure to the first gas source port, the operation that the sealing oil in the first oil cavity is transferred into the cracking tube can be realized, the lysate is sealed, and aerosol pollution is prevented;
when the first mechanical rotary valve is at the third position, the first gas source channel and the second gas source channel, and the first lysate transfer channel and the second lysate transfer channel are in an open state; the first oil seal channel and the second oil seal channel are still in a cut-off state, at the moment, the first air source port, the first air source channel, the first valve inner air source channel, the second air source channel, the cracking tube, the first cracking tube transfer channel, the first valve inner cracking liquid channel, the second cracking tube transfer channel, the nucleic acid extraction cavity port and the atmosphere port of the third mechanical rotary valve are communicated, at the moment, an external power source provides power to give positive pressure to the first air source port, and then the operation that the cracking liquid in the cracking tube is transferred into the nucleic acid extraction cavity can be realized;
when the second mechanical rotary valve is at the first position, the first eluent transfer channel and the second eluent transfer channel, the third oil seal channel and the fourth oil seal channel, and the third air source channel and the fourth air source channel are in a turn-off state;
when the second mechanical rotary valve is at the second position, the first eluent transfer channel, the second eluent transfer channel, the third gas source channel and the fourth gas source channel are in an open state; the third oil seal channel and the fourth oil seal channel are still in a shut-off state, at the moment, the third air source port, the third air source channel, the second valve inner air source channel, the fourth air source channel, the amplification tube, the second eluent transfer channel, the first valve inner eluent transfer channel, the first eluent transfer channel, the nucleic acid extraction cavity port and the third mechanical rotary valve belong to the atmosphere port are communicated, at the moment, the power source is externally connected to provide power to give negative pressure to the third air source port, and the operation that the eluent in the nucleic acid extraction cavity is transferred into the amplification tube can be realized;
when the second mechanical rotary valve is at a third position, the third oil seal channel and the fourth oil seal channel, and the third air source channel and the fourth air source channel are in a communicated state; the first eluent transfer channel and the second eluent transfer channel are in a cut-off state, at the moment, the third air source port, the third air source channel, the second valve inner air source channel, the fourth air source channel, the amplification tube, the fourth oil seal channel, the second valve inner oil seal channel, the third oil seal channel, the second oil cavity and the second oil cavity port are communicated with each other, at the moment, an external power source provides power to give negative pressure to the third air source port, the operation that sealing oil in the second oil cavity is transferred to the amplification tube can be achieved, amplification reagents are sealed, and aerosol pollution is prevented.
6. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the top of the card box body is provided with a mechanical rotary valve, namely a third mechanical rotary valve;
a third rotating rod mounting position, a second air source port and an atmospheric port are arranged in the third mechanical rotary valve;
the third mechanical rotary valve takes the port of the nucleic acid extracting cavity as the center to carry out rotary motion;
when the third mechanical rotary valve is positioned at the first position, the atmosphere port to which the third mechanical rotary valve belongs is always concentrically aligned with the port of the nucleic acid extraction cavity, and in this state, the second gas source port is in a turn-off state with the port of the waste liquid cavity to which the waste liquid cavity belongs, the port of the waste liquid cavity to which the mixing cavity belongs, the port of the first cleaning liquid storage cavity to which the first cleaning liquid storage cavity belongs, the port of the second cleaning liquid storage cavity to which the second cleaning liquid storage cavity belongs, and the port of the eluent storage cavity to which the eluent storage cavity belongs;
when the third mechanical rotary valve is at the second position, the second air source port is communicated with the mixing cavity port, the mixing cavity, the mixing channel, the nucleic acid extraction cavity port and the atmosphere port which the mixing cavity belongs to; at the moment, the second gas source port and the waste liquid cavity port belonging to the waste liquid cavity, the first cleaning liquid storage cavity port belonging to the first cleaning liquid storage cavity, the second cleaning liquid storage cavity port belonging to the second cleaning liquid storage cavity and the eluent storage cavity port belonging to the eluent storage cavity are in a turn-off state, and at the moment, the external power source provides alternate positive pressure and negative pressure to the second gas source channel, so that the mixing operation of the reagent in the nucleic acid extraction cavity and the mixing cavity can be realized;
when the third mechanical rotary valve is at a third position, the second gas source port is communicated with the waste liquid cavity port to which the waste liquid cavity belongs, the waste liquid cavity, the waste liquid transfer channel, the nucleic acid extraction cavity port and the atmosphere port; at the moment, the second gas source port and the port of the waste liquid cavity to which the mixing cavity belongs, the port of the first cleaning liquid storage cavity to which the first cleaning liquid storage cavity belongs, the port of the second cleaning liquid storage cavity to which the second cleaning liquid storage cavity belongs and the port of the eluent storage cavity to which the eluent storage cavity belongs are in a turn-off state, and at the moment, the external power source provides negative pressure to the second gas source channel, so that the transfer operation of the waste liquid in the nucleic acid extraction cavity can be realized;
when the third mechanical rotary valve is positioned at the fourth position, the second gas source port is communicated with a first cleaning solution storage cavity port, a first cleaning solution storage cavity, a first Z-shaped reagent injection channel, a nucleic acid extraction cavity port and an atmosphere port which the first cleaning solution storage cavity belongs to; at the moment, the second gas source port is in a turn-off state with the waste liquid cavity port to which the waste liquid cavity belongs, the mixing cavity port to which the mixing cavity belongs, the second cleaning liquid storage cavity port to which the second cleaning liquid storage cavity belongs, and the eluent storage cavity port to which the eluent storage cavity belongs, and at the moment, the external power source provides positive pressure to the second gas source channel, so that the operation of injecting the first cleaning liquid in the first cleaning liquid storage cavity into the nucleic acid extraction cavity can be realized;
when the third mechanical rotary valve is positioned at the fifth position, the second gas source port is communicated with a second cleaning solution storage cavity port to which the second cleaning solution storage cavity belongs, the second cleaning solution storage cavity, the second Z-shaped reagent injection channel, the nucleic acid extraction cavity port and the atmosphere port; at the moment, the second gas source port and the waste liquid cavity port belonging to the waste liquid cavity, the mixing cavity port belonging to the mixing cavity, the first cleaning liquid storage cavity port belonging to the first cleaning liquid storage cavity and the eluent storage cavity port belonging to the eluent storage cavity are in a turn-off state, and at the moment, the external power source provides positive pressure to the second gas source channel, so that the operation of injecting the second cleaning liquid in the second cleaning liquid storage cavity into the nucleic acid extraction cavity can be realized;
when the third mechanical rotary valve is at a sixth position, the second gas source port is communicated with an eluent storage cavity port, an eluent storage cavity, a third Z-shaped reagent injection channel, a nucleic acid extraction cavity port and an atmosphere port which the eluent storage cavity belongs to; at this time, the second gas source port is in a shut-off state with the waste liquid chamber port to which the waste liquid chamber belongs, the mixing chamber port to which the mixing chamber belongs, the first cleaning liquid storage chamber port to which the first cleaning liquid storage chamber belongs, and the second cleaning liquid storage chamber port to which the second cleaning liquid storage chamber belongs, and at this time, the external power source provides positive pressure to the second gas source channel, so that the operation of injecting the eluent in the eluent storage chamber into the nucleic acid extraction chamber can be realized.
7. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the bottom of the card box body is provided with two trapezoidal bosses, namely a first trapezoidal boss and a second trapezoidal boss, and the two trapezoidal bosses are used for sealing and installing the cracking tube and the amplification tube.
8. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the card box body top is provided with three air supply port, first air supply port, second air supply port, third air supply port promptly, and the air supply port inserts the structure of card box as external air supply, can accept the air supply power that comes from peristaltic pump, syringe pump, vacuum generator and generate.
9. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the second air source channel, the second oil seal channel and the first lysate transfer channel are arranged on the first trapezoid boss at the bottom of the card box body, inserted into the lysate tube and respectively used as a liquid reagent transfer channel and an air source power supply channel; the bottom of the card box body is provided with a second eluent transfer channel, a fourth oil seal channel and a fourth gas source channel on a second trapezoidal boss; inserted into the interior of the amplification tube for serving as a liquid reagent transfer and a gas source power supply channel, respectively.
10. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the cracking tube and the amplification tube are arranged at the bottom of the card box body, and a power source is arranged at the top of the card box body and used for providing power for transferring reagents in the card box.
11. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the cracking tube is used for high-temperature cracking of a sample, and a dry powder reagent of a cracking solution is pre-embedded in advance;
the amplification tube is used for nucleic acid adsorption and amplification detection, and amplification liquid dry powder reagents are pre-buried in advance;
the first cleaning liquid storage cavity is pre-embedded with first cleaning liquid;
and a second cleaning liquid is pre-embedded in the second cleaning liquid storage cavity.
12. The closed microfluidic nucleic acid detection cartridge of claim 1, wherein:
the method is used for separating DNA and RNA from a biological sample and directly completing nucleic acid amplification detection in an amplification tube.
13. An integrated nucleic acid extraction, amplification and detection method is characterized by comprising the following steps:
step 1) preparation step: adding a certain volume of suspension into a clinical sample to resuspend a swab sample;
step 2) preparation step: injecting the sample obtained in the preparation step with a certain volume into a cracking tube, fully mixing the sample with a dry powder reagent of a cracking solution in the cracking tube, then installing the cracking tube on a first trapezoidal boss of a closed card box, and installing an amplification tube on a second trapezoidal boss in advance;
step 3) preparation step: the method comprises the following steps of placing a card box body in an integrated nucleic acid detector, ensuring that a cracking tube and an amplification tube are inserted into a heating module in an aligned mode, and completing correct installation of the whole card box;
step 4) lysis of the sample, release of nucleic acids: the driving mechanism in the integrated nucleic acid detector and the first rotating rod which the first mechanical rotating valve belongs to are arranged at positions, the first mechanical rotating valve is controlled to rotate to a second position, an external power source provides power to provide negative pressure for the first air source port, so that the operation of transferring sealing oil in the first oil cavity into the cracking tube can be realized, a sample to be cracked in the cracking tube is sealed, aerosol pollution in the cracking process is prevented, then, the heating process is started, the sample in the cracking tube is fully cracked, and nucleic acid is released as much as possible;
step 5), transferring the lysate into a nucleic acid extraction cavity: the driving mechanism in the integrated nucleic acid detector and the first rotating rod which the first mechanical rotating valve belongs to are arranged at positions for controlling the first mechanical rotating valve to rotate to a third position, and an external power source provides power to provide positive pressure for the first air source port, so that the operation of transferring the lysate in the lysis tube into the nucleic acid extraction 8 can be realized;
step 6) reagent mixing, nucleic acid adsorption: a driving mechanism in the integrated nucleic acid detector is connected with a third rotating rod which a third mechanical rotating valve belongs to, the third mechanical rotating valve is controlled to rotate to a second position, an external power source provides alternate positive pressure and negative pressure for a second air source channel, the mixing operation of a nucleic acid extraction cavity and a reagent in a mixing cavity can be realized, and magnetic bead dry powder pre-buried in the nucleic acid extraction cavity is fully mixed with lysis solution so as to be convenient for fully adsorbing nucleic acid;
step 7), a waste liquid transferring step I: firstly, placing a magnet at the bottom of a nucleic acid extraction cavity; then, a driving mechanism in the integrated nucleic acid detector is connected with a third rotating rod installation part to which a third mechanical rotating valve belongs, the third mechanical rotating valve is controlled to rotate to a third position, and an external power source provides negative pressure to a second air source channel, so that waste liquid in the nucleic acid extraction cavity can be transferred into the waste liquid cavity;
step 8) magnetic bead washing step a: firstly, removing the magnet, connecting a driving mechanism in the integrated nucleic acid detector with a third rotating rod which a third mechanical rotating valve belongs to, controlling the third mechanical rotating valve to rotate to a fourth position, and providing positive pressure to a second air source channel by an external power source, so that the operation of injecting the first cleaning solution in the first cleaning solution storage cavity into the nucleic acid extraction cavity can be realized; subsequently, repeating the step 6) to ensure that the cleaning solution can sufficiently clean the magnetic beads;
step 9) waste liquid transfer step two: repeating step 7);
step 10) magnetic bead washing step B: firstly, removing a magnet, connecting a driving mechanism in the integrated nucleic acid detector with a third rotating rod mounting part to which a third mechanical rotating valve belongs, controlling the third mechanical rotating valve to rotate to a fifth position, providing positive pressure to a second air source channel by an external power source, realizing the operation of injecting a second cleaning solution in a second cleaning solution storage cavity into a nucleic acid extraction cavity, and then, repeating the step 6) to ensure that the cleaning solution can fully clean magnetic beads;
step 11) waste liquid transfer step three: repeating step 7);
step 12) nucleic acid elution step: firstly, removing the magnet, connecting a driving mechanism in the integrated nucleic acid detector with a third rotating rod which a third mechanical rotating valve belongs to at the installation position, controlling the third mechanical rotating valve to rotate to a sixth position, and providing positive pressure to a second gas source channel by an external power source, so that the operation of injecting the eluent in the eluent storage cavity into the nucleic acid extraction cavity can be realized; subsequently, repeating the step 6) to ensure that the eluent is fully contacted with the magnetic beads and is uniformly mixed with the magnetic beads;
step 13) transfer of the eluent: a driving mechanism in the integrated nucleic acid detector is connected with a second rotating rod mounting part of a second mechanical rotating valve, the second mechanical rotating valve is controlled to rotate to a second position, an external power source provides power to supply negative pressure to a third gas source port, so that the operation that eluent in a nucleic acid extraction cavity is transferred into an amplification tube can be realized, and after the nucleic acid eluent enters the amplification tube, the nucleic acid eluent is fully mixed with an amplification liquid dry powder reagent in the amplification tube to prepare for downstream nucleic acid amplification reaction;
step 14) preparation of amplification reaction: a driving mechanism in the integrated nucleic acid detector is connected with a second rotating rod which the second mechanical rotating valve belongs to, the second mechanical rotating valve is controlled to rotate to a third position, an external power source provides power to supply negative pressure to a third gas source port, the operation that sealing oil in the second oil cavity is transferred to enter an amplification tube is realized, amplification reagents are sealed, and aerosol pollution is prevented;
step 15) amplification detection: the driving mechanism in the integrated nucleic acid detector is connected with the second rotating rod which the second mechanical rotating valve belongs to, and the second mechanical rotating valve is controlled to rotate to the first position, so that the amplification tube is isolated from the outside, and aerosol pollution is prevented.
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| CN202010863164.2A CN111909835A (en) | 2020-08-25 | 2020-08-25 | Closed micro-fluidic nucleic acid detection card box |
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Application publication date: 20201110 |