CN116731843A - Visual instant nucleic acid detection system and use method - Google Patents
Visual instant nucleic acid detection system and use method Download PDFInfo
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- CN116731843A CN116731843A CN202310976299.3A CN202310976299A CN116731843A CN 116731843 A CN116731843 A CN 116731843A CN 202310976299 A CN202310976299 A CN 202310976299A CN 116731843 A CN116731843 A CN 116731843A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a visual instant nucleic acid detection system and a use method thereof, belonging to the technical field of microfluidics. The nucleic acid detection system comprises a fully integrated microfluidic chip and a heating module, wherein the microfluidic chip comprises a substrate, a cover plate and a slide plate, the heating module is powered by a mobile power supply, and the graphene heating plate provides stable temperature. The microfluidic chip structure comprises a nucleic acid extraction region, a nucleic acid mixing region and a nucleic acid amplification region. The channels are respectively communicated with the reaction tanks and the vent holes; the nucleic acid extraction area is used for introducing a sample, and cracking and eluting target nucleic acid; the nucleic acid mixing area is used for fully mixing the purified nucleic acid with the amplification solution; the nucleic acid amplification zone has a plurality of reaction cells, and each reaction cell is physically isolated by means of a slide plate. The instant detection system provided by the invention can realize the whole process of automatic detection, is simple and convenient to operate, has high flux, and can be widely applied to the fields of personalized medicine, species identification and the like.
Description
Technical Field
The invention belongs to the technical field of microfluidics, and relates to a visual full-integrated instant nucleic acid detection system and a use method thereof.
Background
The microfluidic chip is a chip integrating basic functional units such as a micrometer-scale channel, a reaction tank and the like on an operation platform, and is used in the technical fields of nucleic acid detection, cell culture and the like in the biological, chemical and medical fields. The micro-scale control of the flow of a small amount of liquid is realized through structures such as micro-channels, micro-pumps, micro-valves and the like which are precisely processed on the chip.
Traditional nucleic acid detection requires the following steps: and (3) a step of: pretreating a detection sample, and removing impurities interfering with detection in the sample to obtain a sample to be detected; and II: the nucleic acid is required to be cracked, washed and eluted for the sample to be detected, so as to realize the purification of the nucleic acid; thirdly,: mixing the purified nucleic acid molecules with an amplification reagent to obtain a solution to be amplified; fourth, the method comprises the following steps: performing nucleic acid amplification reaction by using a special nucleic acid amplification instrument; fifth step: and carrying out analysis and detection on the sample result. The whole nucleic acid detection process has high professional requirements for laboratory staff, long time and needs large-volume equipment and a special laboratory for operation. The university of Islamic, atazade, zahra Noormohammadi, teaches a highly efficient method for detecting transgenic rice, soybean and maize element-specific Polymerase Chain Reaction (PCR). They developed PCR detection methods with a detection limit of 0.25% of transgenic element content in the sample and showed reliable results even when very complex matrices such as processed foods, lecithins and oils were used as starting materials. However, they are only used in laboratory detection of transgenic rice, soybean, etc., and all of nucleic acid extraction, washing, and mixing require laboratory equipment and clean environments, and cannot be performed in real-time on-site detection.
The visual nucleic acid detection system integrates basic functions of nucleic acid extraction, cleaning, mixing, amplification, detection and the like on a chip with smaller volume, and eliminates large equipment and instruments in the traditional nucleic acid detection process. The chip is hermetically packaged in the detection process, the flow of the liquefied sample is controlled through simple operation, the whole step of nucleic acid detection is completed, the detection result can be visually observed, the professional requirement on personnel is reduced, and impurities can be effectively prevented from entering the reaction tank to influence the detection result.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a visual instant nucleic acid detection system and a use method thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
a visual instant nucleic acid detection system comprises a fully integrated sliding microfluidic chip and a heating module. The fully-integrated sliding microfluidic chip is used for sample splitting, nucleic acid extraction and purification, nucleic acid and amplification solution mixing and nucleic acid amplification, and consists of a substrate 1, a cover plate 2 and a slide plate 3, wherein the substrate 1 is respectively attached to the cover plate 2 and the slide plate 3. The heating module is used for heating the chip at constant temperature so as to enable the amplification reaction to be carried out normally.
The upper surface of the chip substrate 1 is provided with a plurality of grooves with different shapes; the cover plate 2 is used for being attached to the substrate 1, after the attachment, a groove on the substrate 1 and a gap between the cover plate 2 form each chamber, each formed structure is sequentially an air inlet hole 4, a micro-channel 5, a cracking pool 6, a cleaning pool A7, a cleaning pool B8, an elution pool 9, an amplification reagent pool 10, a mixing pool 11, an exhaust hole A12, a plurality of nucleic acid amplification reaction pools 13 and a waste liquid pool 14 along the flowing direction of liquid, and an exhaust hole B15 is arranged above the waste liquid pool 14; the cover plate 2 is designed to expose a plurality of nucleic acid amplification reaction tanks 13, does not completely cover the area of the substrate 1, realizes the integral connection of the cover plate 2 at one side of the area, is only attached to a small part of the substrate 1 in the area, aims to attach the slide plate 3 to the substrate 1 through the exposed area, seals the area of the nucleic acid amplification reaction tanks 13, and therefore forms a flow channel among the amplification reaction tanks before the slide plate 3 slides, is communicated with each reaction tank, and seals and mutually independent the amplification reaction tanks after sliding; the sliding sheet 3 is of a reverse-step double-layer structure, the lower layer structure is attached to the substrate 1, the widths of the upper layer structure and the lower layer structure are the same, the lengths of the upper layer structure and the cover sheet 2 are different, the length of the lower layer structure is the same as the length of the gap of the cover sheet 2 corresponding to the exposed area of the plurality of nucleic acid amplification reaction tanks 13, the width of the lower layer structure is smaller than the width of the cover sheet 2, and the length of the upper layer structure is slightly larger than the length of the lower layer structure, so that a reverse-step structure is formed, and the sliding on the substrate 1 is realized; the sliding plate 3 is integrally processed, a row of circular grooves 16 and a row of square grooves 17 are formed in the contact surface of the lower structure, which is attached to the substrate 1, one row of square grooves 17 are used for being matched with a plurality of nucleic acid amplification reaction tanks 13 on the substrate 1 to form a micro-channel according to the sliding direction, liquid can be smoothly injected into the nucleic acid amplification reaction tanks 13 from the mixing tank 11, one row of circular grooves 16 are used for being matched with the nucleic acid amplification reaction tanks 13 on the substrate 1 to form independent amplification reaction tanks, so that the nucleic acid amplification reaction tanks 13 can detect a plurality of different target genes, and a stringing phenomenon can not be generated.
The heating module comprises a main control cabin 18, an amplification cabin 19, a sliding plate 20, a mounting plate 21 and a battery cabin 22. The main control bin 18 is used for placing a temperature control module 23; the amplification bin 19 is used for placing chips so as to enable the chips to be in a constant-temperature state for amplification reaction; the sliding plate 20 is used for sealing the amplification bin 19 to form a closed space, so that heat loss is reduced; the mounting plate 21 is fixedly connected with the amplification bin 19 in a T-shaped groove mode; the battery compartment 22 is used for placing a portable power source for supplying power.
Specific:
the air inlet hole 4 is used for connecting a syringe, and the liquid is pushed into the appointed cavity by a pneumatic mixing mode.
The micro flow channel 5 is used for connecting each chamber, so that liquid can smoothly enter each chamber.
The three chambers of the cracking tank 6, the cleaning tank A7 and the cleaning tank B8 and a runner communicated with the three chambers are called a nucleic acid extraction area and are used for realizing nucleic acid cracking and nucleic acid purification. Sample injection holes are respectively arranged at the tips of the cover plate 2 corresponding to the cracking pool 6, the cleaning pool A7 and the cleaning pool B8. The sample injection hole is used for introducing the pyrolysis liquid, the cleaning liquid A and the cleaning liquid B into the corresponding reaction tanks. The lysis cell 6 is used for releasing nucleic acid molecules in cells, and the washing cell A7 and the washing cell B8 are used for washing out impurities stained on the surfaces of the nucleic acid molecules.
The three chambers of the elution pool 9, the amplification reagent pool 10 and the mixing pool 11 are called a nucleic acid mixing area and are used for realizing the mixing of nucleic acid molecules and amplification reagents to form a solution to be amplified. The three chambers are kept in a communicated state, wherein an exhaust hole A12 is arranged at the position of the cover plate 2 corresponding to the mixing tank 11, and the elution tank 9 and the amplification reagent tank 10 are respectively provided with sample injection holes. The sample inlet is used for introducing eluent and amplification reagent into the corresponding reaction tank, and the exhaust hole A12 is used for discharging the gas in the mixing tank 11 so as to mix the liquid in the mixing tank 11.
The plurality of nucleic acid amplification reaction tanks 13 are referred to as nucleic acid amplification regions for performing nucleic acid amplification by a solution to be amplified containing nucleic acid molecules. The solution to be amplified enters the nucleic acid amplification region from one end of the mixing tank 11, sequentially fills up the plurality of amplification reaction tanks 13, and the remaining liquid flows into the waste liquid tank 14.
The waste liquid pool 14 is used for storing redundant liquid, and is provided with an exhaust hole B15 for exhausting gas in the nucleic acid amplification area so that the liquid to be reacted fills all the nucleic acid amplification reaction pools 13.
The long side of the sliding plate 3 is aligned and attached to one side, which is not attached to the cover plate 2, of the plurality of nucleic acid amplification reaction cells 13 on the substrate 1, one side, which is provided with square grooves 17 and round grooves 16, of the sliding plate 3 is attached to the plurality of nucleic acid amplification reaction cells 13 on the substrate 1, the left end and the right end of the lower structure of the sliding plate 3 are aligned with corresponding gaps of the cover plate 2, so that the sliding plate 3 is tightly attached to the substrate 1 and the cover plate 2, and at the moment, the square grooves 17 on the side, which is contacted with the substrate 1, of the sliding plate 3 are combined with the substrate 1 to form a flow channel, and liquid flow can be realized; the slide plate 3 is pushed to the attached side of the substrate 1 and the cover plate 2 on the areas of the plurality of nucleic acid reaction tanks 13 along the longitudinal direction of the chip, the slide plate 3 is displaced to the overlapping position of the circular groove 16 of the slide plate 3 and the plurality of nucleic acid amplification reaction tanks 13, at the moment, the circular groove 16 of the slide plate 3 on the surface contacted with the substrate 1 and the plurality of nucleic acid amplification reaction tanks 13 are combined into a plurality of independent nucleic acid amplification reaction tanks, and the phenomenon of hole crossing does not occur between the two. The sliding plate 3 is sealed and fixed with the substrate 1 in a paraffin sealing mode.
Furthermore, the cracking tank 6, the cleaning tank A7 and the cleaning tank B8 are square with one end pointed cone and one end, the three reaction tanks are arranged in parallel, and the square ends of the three reaction tanks are connected with the three chambers by adopting the micro-channel 5. The micro-channel 5 is used for transfer of magnetic beads.
Further, the elution pool 9 and the amplification reagent pool 10 have a depth drop, and the sample injection hole of the amplification reagent pool 10 is arranged on one side of the amplification reagent pool 10 close to the elution pool 9, and the liquid injected from the sample injection hole flows to the other end with small flow resistance.
Furthermore, the base sheet 1, the cover sheet 2 and the sliding sheet 3 can be machined and formed, and the materials are polycarbonate PC; injection molding may be used, and the material may be cycloolefin copolymer. Preferably, the base sheet 1, the cover sheet 2 and the slider 3 are made of plastic (polycarbonate or COC).
Furthermore, the substrate 1 and the cover plate 2 are bonded by adhesive, and the substrate 1 and the slider 3 are sealed by paraffin.
Further, the substrate 1 is a strip plate structure, and the thickness of the substrate is 2.5-10 mm; the cover plate 2 is of a strip plate-shaped structure, and the thickness of the cover plate is 0.1-5 mm; the sliding piece 3 is of a strip plate structure, and the thickness of the sliding piece is 0.2-5 mm. The depths of the cracking pool 6, the cleaning pool A7 and the cleaning pool B8 are 1-2 mm; the cross-sectional dimension of the microchannel 5 structure is not less than 1x1mm. The depth of the elution pool 9 is 0.2-0.8 mm; the depth of the amplification reagent pool 10 and the mixing pool 11 is 1.5-3 mm; the depth of the plurality of nucleic acid amplification reaction tanks 13 is not less than 1mm. Further, the size of the master control bin 18 is not smaller than 80×80×25mm (length×width×height), the size of the amplification bin 19 is not smaller than 160×80×25mm, and the size of the battery bin 22 is not smaller than 235×80×40mm.
Further, the number of the nucleic acid amplification reaction tanks 13 is 16, the number can be adjusted according to practical situations, and different primers can be added into each nucleic acid amplification reaction tank to detect different targets.
Furthermore, the air inlet 4 is connected with a disposable injector, which is used for fully mixing the nucleic acid and the amplification solution, so as to ensure the accuracy of the amplification result.
Further, the cracking tank 6 is provided with a cracking liquid such as: the Vazyme RM101-AB and the cleaning solution in the cleaning tank A7 are placed with, for example: the Vazyme RM101-AD and the cleaning tank B8 are filled with cleaning liquid such as: the eluent is placed in the Vazyme RM101-AE, elution pool 9, for example: vazyme RM101-AF, amplification reagent pool 10 holds a mixture such as:LAMP Kit。
further, the heating module comprises a main control cabin 18, an amplification cabin 19, a sliding plate 20, a mounting plate 21, a battery cabin 22 and a temperature control module 23. The main control cabin 18 is divided into two layers, the upper half space is used for placing the temperature control module 21, the lower half space is used for placing wires, and a power switch is arranged in the main control cabin 18, namely, a circular through hole is formed in the uppermost part of the main control cabin 18 and used for exposing an LED display screen and a control switch of the temperature control module 23. The middle partition plate of the main control cabin 18 is provided with a semicircular structure for fixing the temperature control module 23 in the upper half space. The bottom of the main control cabin 18 is provided with a square through hole for battery connection. The amplification bin 19 is divided into three layers: the upper layer and the lower layer are used for placing the graphene heating plate, and the heat is guaranteed to be uniform by adopting the heat-insulating rock wool plate and the aluminum foil, so that heat loss is reduced. The middle layer space is used for placing a fully integrated sliding microfluidic chip. The battery compartment 22 is closed at one end and open at one end, a movable power supply is arranged in the battery compartment, and cube protrusions are arranged at four corners of the contact surface of the battery compartment and other compartments, and are used for fixing the positions of the main control compartment 18 and the amplification compartment 19. The mounting plate 21 is provided with a T-shaped protrusion on one side thereof. The other surface of the expansion chamber is used for being matched with the edge of the inner upper surface of the battery chamber 22, and the T-shaped bulge of the expansion chamber is used for being matched with a T-shaped groove at the bottom of the expansion chamber 19, so that the connection and the fixation between the battery chamber 22 and the expansion chamber 19 are realized; and a square cover plate is arranged at one end of the mounting plate 21 and is used for sealing the battery compartment 22.
Further, the main control cabin 18, the amplification cabin 19, the mounting plate 21 and the battery cabin 22 are assembled by adopting a splicing method. The main control bin 18 and the amplification bin 19 are matched and fixed in a mortise and tenon joint mode. Two convex ends are arranged at one end of the main control bin 18, and a groove is arranged at one end of the amplification bin 19, so that the matching and fixing are realized.
Further, the heating module is made of a resin material, for example: ABS resin material.
Further, the size of the main control cabin 18 is not smaller than 80x80x25mm (length x width x height); the size of the amplification bin 19 is not smaller than 160x80x25mm; the thickness of the sliding plate 20 is 0.5-5 mm; the thickness of the mounting plate 21 is 1-6 mm; the battery compartment 22 is sized no smaller than 235x80x40mm.
A method of using a visual instant nucleic acid detection system, comprising the steps of:
step 1: paraffin and different primers are added to each of the plurality of nucleic acid amplification reaction tanks 13, and the mixture is air-dried at room temperature until the liquid paraffin solidifies.
Step 2: sequentially adding the lysate, the cleaning solution A and the cleaning solution B into the corresponding reaction tanks, then injecting the eluent into the elution tank 9, and finally injecting the amplification reagent into the amplification reagent tank 10.
Step 3: after each reagent was injected, the beads were injected into the lysis cell 6. And sealing all the sample injection holes by using a sealing adhesive tape to ensure that the liquid does not move any more. The magnetic beads need to be soaked in a reagent to be detected in advance, so that the surfaces of the magnetic beads can be specifically combined with nucleic acid to be detected.
Step 4: and moving the magnetic beads by using a magnetic rod to sequentially pass through a cracking pool 6, a cleaning pool A7, a cleaning pool B8 and an eluting pool 9, wherein after the magnetic beads stay in each reaction pool, the nucleic acid molecules adsorbed on the magnetic beads are eluted in the eluting pool 9, so as to obtain the eluent of the target nucleic acid. And then the magnetic beads are moved back to the cleaning pool B8, so that the magnetic beads are ensured not to influence the subsequent mixing reaction, and finally, the eluent for mixing the target nucleic acid is obtained in the eluting pool 9.
Step 5: the gas is squeezed by a disposable syringe so that the eluent in the elution tank 9 of step 4 flows through the amplification reagent tank 10 to the mixing tank 11 even if the mixed solution containing nucleic acid and amplification reagent enters the mixing tank 11, and the mixing is completed by repeated suction a plurality of times. The slide plate 3 is covered, so that the positions of a row of square grooves 17 on the contact surface of the slide plate 3 and the base surface 1 correspond to the positions of a plurality of nucleic acid amplification reaction tanks 13 one by one, and at the moment, the side edges of the slide plate 3 are aligned with the side edges of the opening of the cover plate 2. The syringe is pressed again to fill the micro-channels formed by the mixed solution in the mixing tank 11 and the nucleic acid amplification reaction tanks 13 through the square grooves 17 of the slide plate 3 with the nucleic acid amplification reaction tanks 13 in sequence until part of the liquid enters the waste liquid tank 14.
Step 6: and then moving the slide plate 3, and sliding the slide plate 3 along the chip to the position where a row of circular grooves 16 of the slide plate 3 are overlapped with the plurality of nucleic acid amplification reaction tanks 13, wherein the plurality of nucleic acid amplification reaction tanks 13 are mutually independent. The chip is placed in the middle layer space of an amplification bin 19 of a self-made heating module, the temperature control module 23 is clicked to start heating, the temperature is raised to 60-65 ℃, after paraffin in the nucleic acid amplification reaction tank 13 is melted, the primer in the nucleic acid amplification reaction tank and target nucleic acid to be detected are subjected to amplification reaction, after a few minutes, the color change is visually seen, and the color change is a positive target, so that a detection result is obtained.
Further, the residence time of each reaction tank in the step 4 is 1 minute; the reaction time in step 5 is less than 40 minutes.
The invention simplifies the nucleic acid extraction and purification steps into the steps that magnetic beads move in each reaction tank and are mixed with liquid by adopting a syringe. The chip adopts magnetic rod control and injector control, and realizes the process of sample sealing detection.
The beneficial effects of the invention are as follows:
(1) The number of detection targets is large. The invention can detect at most 16 target primers simultaneously, realizes single-sample multi-target detection, improves the detection efficiency and reduces the operation difficulty of detection personnel.
(2) High integration and visual results. The system integrates the whole process of nucleic acid detection, including nucleic acid extraction, mixing, amplification and result observation, simplifies the operation flow, reduces the operation equipment and expands the application range.
Drawings
FIG. 1 is a front view of a microfluidic chip based on a visual instant nucleic acid detection system of the present invention.
FIG. 2 is a schematic structural diagram of a microfluidic chip based on a visual instant nucleic acid detection system according to the present invention.
FIG. 3 is a flow chart showing the independent operation of the amplification reaction cell 13 of the microfluidic chip based on the visual instant nucleic acid detection system of the present invention.
FIG. 4 is an isometric view of a heating module of the present invention based on a visual instant nucleic acid detection system.
FIG. 5 is a schematic exploded view of a heating module based on a visual instant nucleic acid detection system and a perspective view of a master control cartridge 18 of the present invention.
In the figure: 1, a substrate; 2, cover plate; 3, sliding sheets; 4, an air inlet hole; 5 micro-channels; 6, a cracking pool; 7, cleaning the pool A;8, cleaning the pool B;9, eluting a pool; 10 amplification reagent pool; 11 a mixing tank; 12 exhaust holes A;13 multiple nucleic acid amplification reaction cells; 14 a waste liquid pool; 15 exhaust holes B;16 circular grooves; 17 square grooves; 18, a main control bin; 19 amplification bins; 20 sliding plates; a 21 mounting plate; 22 battery bins; 23 temperature control module.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
The methods used in the following technical schemes are conventional methods unless otherwise specified. Materials and reagents used in the following schemes are commercially available.
Referring to fig. 1-5, a visual instant nucleic acid detection system and method of use of the present embodiment includes a fully integrated sliding microfluidic chip and a heating module.
The fully-integrated sliding microfluidic chip part comprises a substrate 1, a cover plate 2 and a slide plate 3, wherein the substrate, the cover plate 2 and the slide plate 3 are all machined and formed in a mode, and the material is polycarbonate PC. The thickness of the base sheet 1 is 3mm, the thickness of the cover sheet 2 is 0.5mm, the thickness of the sliding sheet 3 is 1mm, and the base sheet 1 and the cover sheet 2 are combined in a chemical double-sided adhesive bonding mode.
The heating module comprises a main control cabin 18, an amplification cabin 19, a sliding plate 20, a mounting plate 21 and a battery cabin 22, wherein a 3D printing forming mode can be adopted, the material is acrylonitrile-butadiene-styrene (ABS), and the wall thickness is 4mm. The main control bin 18 and the amplification bin 19 are positioned at the same horizontal position, are positioned right above the battery bin 22 and are connected by mortise and tenon joints of the mounting plate 21, and the sliding plate 20 is used for sealing the amplification bin 19 and is positioned at one side of the amplification bin 19.
The substrate 1 is provided with a reaction tank which comprises a cracking tank 6, a cleaning tank A7, a cleaning tank B8, an eluting tank 9, an amplification reagent tank 10, 16 amplification reaction tanks 13 and a waste liquid tank 14. Reagents are placed in the reaction tank, and the reagents comprise a lysate for extracting nucleic acid, a cleaning solution, an eluent and an amplification reagent. And magnetic beads are placed in the cracking pool 6 and are used for extracting nucleic acid. And a primer is placed in the amplification reaction tank 13 and used for detecting a sample to be detected. The cover plate 2 is provided with an air inlet 4, a sample injection hole, exhaust holes A12 and B15, and sample injection is carried out for the reaction tank through the sample injection hole. The air inlet hole 4 is used for providing power support for the solution in the chip to uniformly mix the liquid, one end of the air inlet hole is connected with the chip channel, and the other end of the air inlet hole is provided with an injector; the air inlet 4 is connected with the elution pool 9, the amplification reagent pool 10, the amplification reaction pool 13 and the waste liquid pool 14 in sequence through the micro-channel 5.
In each reaction tank: the cracking pool 6 is internally provided with a cracking liquid Vazyme RM101-AB, a cleaning liquid Vazyme RM101-AD, a cleaning pool A7, a cleaning liquid Vazyme RM101-AE, an eluting pool 9, an eluting liquid Vazyme RM101-AF and a mixed liquid, respectively, the amplification reagent pool 10 is provided with a mixing liquidLAMP Kit. The amplification reaction tank 13 is internally provided with a primer, and the bottom of the tank is sealed by paraffin.
The main control bin 18, the amplification bin 19, the sliding plate 20, the mounting plate 21 and the battery bin 22 are spliced in a mortise and tenon mode.
Based on the nucleic acid detection system, a visual instant nucleic acid detection system and a use method thereof comprise the following steps.
Step 1, paraffin and 16 different primers are respectively added into 16 nucleic acid amplification reaction tanks 13, and the mixture is air-dried at room temperature until liquid paraffin is solidified.
Step 2, adding the lysate, the cleaning solution A and the cleaning solution B into the corresponding reaction tanks in sequence, then injecting the eluent into the elution tank 9, and finally injecting the amplification reagent into the amplification reagent tank 10.
And 3, adding 20 μl of sample into the lysis cell 6 through the sample inlet, wherein the lysis cell 6 contains 2 μl of proteinase K and 2 μl of magnetic beads, and sealing the sample inlet by using a sealing plate.
And 4, carrying out room temperature pyrolysis on the solution in the pyrolysis tank 6 for 5 minutes, moving the magnetic beads to sequentially pass through the pyrolysis tank 6, the cleaning tank A7, the cleaning tank B8 and the elution tank 9, and then staying in each reaction tank for 1 minute, so as to finally elute the nucleic acid adsorbed on the magnetic beads. And then the magnetic beads are moved back to the cleaning pool B8, so that the subsequent amplification reaction is not influenced.
And 5, pushing and pulling the injector, pushing and pressurizing for the first time to mix the solution in the elution pool 9 with the amplification reagent, and repeatedly pushing and pulling for 5 times to fully mix the solution to be detected. The slide sheet 3 is covered so that the positions of the square grooves 17 in the contact surface of the slide sheet 3 with the base surface 1 correspond to the positions of the plurality of nucleic acid amplification reaction cells 13, and at this time, the slide sheet 3 is aligned with the side of the substrate 1 which is not bonded to the cover sheet 2. The syringe was again pushed to pressurize so that the solution to be tested filled up with 16 amplification wells.
And 6, pushing the sliding plate 3 to enable the circular grooves 16 of the sliding plate to coincide with the amplification pools, wherein each amplification pool is independent. And (3) placing the chip in a heating module, heating the chip amplification pool at the constant temperature of 65 ℃, and after reacting for 30 minutes, observing and analyzing with naked eyes, wherein the color of the 4 th amplification reaction pool and the 16 th amplification reaction pool is changed, and the result is positive, so that the result is proved to be a target sample, and the rest reaction pools are negative in the absence of color change.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (9)
1. The visual instant nucleic acid detection system is characterized by comprising a fully integrated sliding microfluidic chip and a heating module; the fully-integrated sliding microfluidic chip is used for sample splitting, nucleic acid extraction and purification, nucleic acid and amplification solution mixing and nucleic acid amplification, and consists of a substrate (1), a cover plate (2) and a sliding plate (3), wherein the substrate (1) is respectively attached to the cover plate (2) and the sliding plate (3); the heating module is used for heating the chip at constant temperature so as to enable the amplification reaction to be normally carried out;
the upper surface of the substrate (1) is provided with a plurality of grooves with different shapes; the cover plate (2) is used for being attached to the substrate (1), after the attachment, gaps between grooves on the substrate (1) and the cover plate (2) form various chambers, a micro-channel (5), a cracking tank (6), a cleaning tank A (7), a cleaning tank B (8), an elution tank (9), a plurality of amplification reagent tanks (10), a mixing tank (11), a plurality of nucleic acid amplification reaction tanks (13) and a waste liquid tank (14) are sequentially arranged along the liquid flow direction, wherein an air inlet hole (4) is formed in the position, located above the initial position, of the cover plate (2) in the micro-channel (5), an exhaust hole A (12) is formed in the position, located above the mixing tank (11), of the cover plate (2), and an exhaust hole B (15) is formed in the position, located above the waste liquid tank (14); different primers can be added into each nucleic acid amplification reaction tank for detecting different targets;
after the cover plate (2) is attached to the substrate (1), an opening is formed in the attaching position of the cover plate (2) corresponding to the nucleic acid amplification reaction tank (13), and the opening is used for placing the slide plate (3);
the slide sheet (3) is of a reverse step type double-layer structure, the lower layer structure is attached to the substrate (1), the upper layer structure is attached to the cover sheet (2), the length of the lower layer is identical to the length of a notch of the exposed area of the plurality of nucleic acid amplification reaction tanks (13) corresponding to the cover sheet (2), the width of the lower layer is smaller than that of the cover sheet (2), and the length of the upper layer is larger than that of the lower layer, so that a reverse step type structure is formed, and the sliding on the substrate (1) is realized.
2. The visual instant nucleic acid detection system according to claim 1, wherein the sliding piece (3) is integrally processed, a row of circular grooves (16) and a row of square grooves (17) are arranged on a contact surface of the lower structure, which is attached to the substrate (1), the row of square grooves (17) are used for being matched with a plurality of nucleic acid amplification reaction tanks (13) on the substrate (1) according to the sliding direction to form micro-channels, liquid can be smoothly injected into the plurality of nucleic acid amplification reaction tanks (13) from the mixing tank (11), and the row of circular grooves (16) are used for being matched with the plurality of nucleic acid amplification reaction tanks (13) on the substrate (1) to form an independent amplification reaction tank, so that the plurality of nucleic acid amplification reaction tanks (13) can detect a plurality of different target genes without generating a serial hole phenomenon.
3. The visual instant nucleic acid detection system of claim 1, wherein the heating module comprises a master control bin (18), an amplification bin (19), a sliding plate (20), a mounting plate (21), and a battery bin (22); the main control bin (18) is used for placing a temperature control module (23); the amplification bin (19) is used for placing a fully-integrated sliding microfluidic chip, so that the chip is in a constant-temperature state for amplification reaction; the sliding plate (20) is used for sealing the amplification bin (19) to form a closed space; the mounting plate (21) is fixedly connected with the amplification bin (19) by adopting a T-shaped groove mode; the battery compartment (22) is used for placing a movable power supply and supplying power.
4. The visual instant nucleic acid detection system according to claim 1, wherein the air inlet (4) is adapted to be connected to a syringe for pushing the liquid into the designated chamber by means of pneumatic mixing.
5. The visual instant nucleic acid detection system of claim 1, wherein:
the micro flow channels (5) are used for connecting the chambers, so that liquid can smoothly enter the chambers;
the three chambers of the cracking pool (6), the cleaning pool A (7) and the cleaning pool B (8) and a runner communicated with the three chambers are called a nucleic acid extraction area and are used for realizing nucleic acid cracking and nucleic acid purification; sample injection holes are respectively arranged at the tips of the cover plate (2) corresponding to the cracking pool (6), the cleaning pool A (7) and the cleaning pool B (8);
the three chambers of the elution pool (9), the amplification reagent pool (10) and the mixing pool (11) are called a nucleic acid mixing area and are used for realizing the mixing of nucleic acid molecules and amplification reagents to form a solution to be amplified; the three chambers are kept in a communicated state, wherein an exhaust hole A (12) is formed in the position, corresponding to the mixing tank (11), of the cover plate (2), and sample injection holes are formed in the eluting tank (9) and the amplifying reagent tank (10) respectively.
6. The visual instant nucleic acid detection system according to claim 1, wherein the base sheet (1) and the cover sheet (2) are connected by means of adhesive bonding; the sliding piece (3) is sealed and fixed with the substrate (1) in a paraffin sealing mode.
7. The visual instant nucleic acid detection system according to claim 1, wherein the substrate (1) is a strip-like structure having a thickness of between 2.5 and 10 mm; the cover plate (2) is of a strip plate-shaped structure, and the thickness of the cover plate is 0.1-5 mm; the sliding piece (3) is of a strip plate-shaped structure, and the thickness of the sliding piece is 0.2-5 mm; the depths of the cracking pool (6), the cleaning pool A (7) and the cleaning pool B (8) are 1-2 mm; the depth of the elution pool (9) is 0.2-0.8 mm; the depth of the amplification reagent pool (10) and the mixing pool (11) is 1.5-3 mm; the depth of the plurality of nucleic acid amplification reaction tanks (13) is not less than 1mm.
8. A method of using the visual instant nucleic acid detection system of any one of claims 1-7, comprising the steps of:
step 1: paraffin and different primers are respectively added into a plurality of nucleic acid amplification reaction tanks (13), and the mixture is air-dried at room temperature until the liquid paraffin is solidified;
step 2: sequentially adding the lysate, the cleaning solution A and the cleaning solution B into the corresponding reaction tanks, then injecting the eluent into the elution tank (9), and finally injecting the amplification reagent into the amplification reagent tank (10);
step 3: after the reagents are injected, the magnetic beads are injected into a cracking pool (6); sealing all the sample injection holes by using a sealing adhesive tape to ensure that liquid does not move any more; the magnetic beads need to be soaked in the reagent to be detected in advance, so that the surfaces of the magnetic beads are adhered with the reagent to be detected;
step 4: moving the magnetic beads by using a magnetic rod, enabling the magnetic beads to sequentially pass through a cracking pool (6), a cleaning pool A (7), a cleaning pool B (8) and an eluting pool (9), and finally eluting nucleic acid molecules adsorbed on the magnetic beads in the eluting pool (9) after the magnetic beads stay in each reaction pool to obtain an eluent of target nucleic acid; then, the magnetic beads are moved back to the cleaning pool B (8), so that the magnetic beads are ensured not to influence the subsequent mixing reaction, and finally, the eluent of the mixed target nucleic acid is obtained in the eluting pool (9);
step 5: extruding gas by using a disposable injector, so that the eluent in the elution pool (9) in the step 4 flows through the amplification reagent pool (10) to reach the mixing pool (11), and even if the mixed solution containing nucleic acid and the amplification reagent enters the mixing pool (11), repeatedly pumping for a plurality of times to finish mixing; covering the sliding sheet (3) so that the positions of a row of square grooves (17) on the contact surface of the sliding sheet (3) and the base surface (1) correspond to the positions of a plurality of nucleic acid amplification reaction tanks (13) one by one, and the side edges of the sliding sheet (3) are aligned with the side edges of the opening of the cover sheet (2); pressing the injector to sequentially fill a plurality of nucleic acid amplification reaction tanks (13) into a micro-channel formed by the mixed solution in the mixing tank (11) and the nucleic acid amplification reaction tanks (13) through square grooves (17) of the sliding plate (3) until part of liquid enters the waste liquid tank (14);
step 6: moving the sliding plate (3), sliding the sliding plate (3) to the corresponding overlapping position of a row of circular grooves (16) of the sliding plate (3) and the plurality of nucleic acid amplification reaction tanks (13) along the chip, wherein the plurality of nucleic acid amplification reaction tanks (13) are mutually independent; the chip is placed in a middle layer space of an amplification bin (19) of a self-made heating module, a switch of a temperature control module (23) is clicked to start heating, the temperature is raised to 60-65 ℃, after paraffin in a nucleic acid amplification reaction tank (13) is melted, an amplification reaction is carried out on the primer in the chip and target nucleic acid to be detected, after a few minutes, the color change is visually seen, and the color change is a positive target, so that a detection result is obtained.
9. The method of claim 8, wherein the residence time of each reaction cell in step 4 is 1 minute; the reaction time in step 5 is less than 40 minutes.
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| CN202310976299.3A CN116731843A (en) | 2023-08-04 | 2023-08-04 | Visual instant nucleic acid detection system and use method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118165804A (en) * | 2024-05-13 | 2024-06-11 | 苏州天隆生物科技有限公司 | Kit, amplification equipment and closed amplification detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118165804A (en) * | 2024-05-13 | 2024-06-11 | 苏州天隆生物科技有限公司 | Kit, amplification equipment and closed amplification detection method |
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