CN115747037A - Nucleic acid analysis card and method of use thereof - Google Patents
Nucleic acid analysis card and method of use thereof Download PDFInfo
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- CN115747037A CN115747037A CN202310024318.2A CN202310024318A CN115747037A CN 115747037 A CN115747037 A CN 115747037A CN 202310024318 A CN202310024318 A CN 202310024318A CN 115747037 A CN115747037 A CN 115747037A
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- nucleic acid
- acid analysis
- analysis card
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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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/14—Thermal energy storage
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid analysis card and a use method thereof, wherein the nucleic acid analysis card comprises a main body, a buckle cover and a phase-change material, wherein the main body is provided with a detection channel, the detection channel comprises a reagent area, a phase-change area and a reaction area which are sequentially communicated, a freeze-dried ball is arranged in the reagent area, the freeze-dried ball comprises an amplification detection reagent, the main body is provided with a sample adding port, and the sample adding port is communicated with the reagent area; the buckle cover is used for opening or closing the sample adding port; the phase-change material is arranged in the phase-change area and forms a liquid seal after being heated and melted. The phase-change material is arranged in the phase-change area, the phase-change material forms a liquid seal after being heated and melted to seal the reaction area, the sealing is more reliable compared with the sealing of a paraffin valve in a solid state, and the liquid seal is re-solidified along with the temperature reduction after the reaction is finished to seal the reaction liquid, so that the loss of the reaction liquid and the cross pollution with the environment, particularly the aerosol pollution, are avoided, and the reliability of the test result is improved.
Description
Technical Field
The invention relates to the field of nucleic acid analysis, in particular to a nucleic acid analysis card and a use method thereof.
Background
At present, in order to improve nucleic acid analysis efficiency and reduce the complexity of nucleic acid analysis equipment, a variety of card-type nucleic acid analysis consumables have appeared. In the prior art, in order to avoid reaction liquid loss caused by high temperature and temperature rise and drop, a reaction area needs to be sealed, and the reaction area is sealed by a flow channel through arranging a solid paraffin valve, but the solid paraffin valve is easy to generate air holes or poor solidification and the like to cause sealing failure.
Disclosure of Invention
The invention mainly aims to provide a nucleic acid analysis card and a using method thereof, which aim to solve the problem that a solid paraffin valve is easy to cause sealing failure.
In order to achieve the purpose, the nucleic acid analysis card provided by the invention comprises a main body, a buckle cover and a phase-change material, wherein the main body is provided with a detection channel, the detection channel comprises a reagent area, a phase-change area and a reaction area which are sequentially communicated, a freeze-dried ball is arranged in the reagent area, the freeze-dried ball comprises an amplification detection reagent, the main body is provided with a sample adding port, and the sample adding port is communicated with the reagent area; the buckle cover is used for opening or closing the sample adding port; the phase-change material is arranged in the phase-change area, and the phase-change material is heated and melted to form a liquid seal.
Optionally, the cover includes a cover body and a skirt, the skirt extends along a radial direction of the cover body, the cover body is used for opening or closing the sample addition port, and the skirt is used for abutting against the main body.
Optionally, the main body includes a sample adding part, the sample adding part is provided with the sample adding port, the skirt is used for abutting against the sample adding part, and the sample adding part protrudes in a direction away from the detection channel so that an installation gap is formed between the skirt and the sample adding part.
Optionally, the body is formed with a stop surface, the stop surface being disposed obliquely, the stop surface being used for stopping the lyophilized pellet from entering the phase transition zone.
Optionally, the main body is formed with a mounting groove, and the phase change material is disposed in the mounting groove.
Optionally, the main body comprises a casing and a sealing membrane which are connected, the sealing membrane is matched with the casing to form the detection channel, the casing is provided with the sample addition port, the sealing membrane is used for being connected with the heating module, and the thickness of the sealing membrane ranges from 0.05mm to 0.2mm.
Optionally, the phase change material is one or a combination of more of solid paraffin with numbers 48, 50, 52, 54, 56, 58, 60, 62 and 64, and the solid paraffin is pharmaceutical grade fully refined paraffin.
Optionally, the phase change material is one or a combination of heneicosane, docosane, tricosane, tetracosane, pentacosane, hexacosane, heptacosane, octacosane, nonacosane and triacontane, and the purity of the phase change material is greater than or equal to 95%.
Optionally, the number of the detection channels is multiple, the detection channels are arranged at intervals, the number of the buckle covers and the number of the phase-change materials are multiple, the buckle covers and the detection channels are arranged in a consistent and one-to-one correspondence manner, and the number of the phase-change materials and the detection channels is consistent and one-to-one correspondence manner.
In addition, the present invention also provides a method for using a nucleic acid analysis card including the nucleic acid analysis card as described above, the method including:
dripping a sample to be detected into the sample adding port;
adjusting the direction of the nucleic acid analysis card to the reagent area downwards, and shaking the nucleic acid analysis card to redissolve the freeze-dried balls to form a reaction solution;
adjusting the direction of the nucleic acid analysis card until the reagent area is upward, and swinging the nucleic acid analysis card to make all reaction liquid fall into the reaction area;
heating the nucleic acid analysis card to melt the phase-change material and make the phase-change material flow to the upper layer of the reaction solution in the reaction area to form a liquid seal;
standing for a preset time within a preset temperature range, and finishing amplification of the reaction liquid within the preset time;
when the standing time reaches the preset time, stopping heating;
when the phase-change material is re-solidified, a nucleic acid analysis result is obtained according to the change of the optical signal in the reaction region.
In the technical scheme of the invention, the detection channel comprises a reagent area, a phase change area and a reaction area which are sequentially communicated from top to bottom. The buckle closure is used for opening or closing the sample adding opening, and the buckle closure opens the sample adding opening, and the sample to be detected enters the reagent area from the sample adding opening. The sample addition port is closed by the buckle cover, so that the detection channel is closed to carry out full reaction, and the reaction liquid is prevented from leaking from the sample addition port. The freeze-drying ball sets up in the reagent district, and the freeze-drying ball includes amplification detection reagent, and the freeze-drying is compared in the stoving, and freeze-drying reagent redissolves and mixing efficiency is higher, and is fit for full component freeze-drying. The reagent area is used for pre-storing and redissolving the freeze-dried spheres, after a sample to be detected is added into the reagent area, the nucleic acid analysis freeze-dried spheres with all components can be redissolved by shaking the nucleic acid analysis card to form reaction liquid, and then the reaction liquid enters the reaction area through the phase change area. The phase-change material forms liquid seal after being heated and melted so as to seal the reaction area, the sealing is more reliable compared with the paraffin valve in a solid state, and the liquid seal is solidified again along with the temperature reduction after the reaction is finished so as to seal the reaction liquid, so that the reliability of the test result is improved. The phase-change material is arranged in the phase-change area, the phase-change material forms a liquid seal after being heated and melted to seal the reaction area, the sealing is more reliable compared with the sealing of a paraffin valve in a solid state, and the liquid seal is re-solidified along with the temperature reduction after the reaction is finished to seal the reaction liquid, so that the loss of the reaction liquid and the cross pollution with the environment, particularly the aerosol pollution, are avoided, and the reliability of the test result is improved.
Drawings
In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the embodiments or technical solutions of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a schematic perspective view of a nucleic acid analysis card according to an embodiment of the present invention;
FIG. 2 is a schematic exploded view of a nucleic acid analysis card according to an embodiment of the present invention;
FIG. 3 is a schematic cross-sectional view of a nucleic acid analysis card according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a plurality of detection channels of a nucleic acid analysis card according to an embodiment of the present invention;
FIG. 5 is a flow chart illustrating a method of use according to an embodiment of the present invention.
The reference numbers indicate:
| reference numerals | Name (R) | Reference numerals | Name(s) |
| 100 | Nucleic |
16 | |
| 10 | |
17 | |
| 11 | |
18 | |
| 111 | |
19 | |
| 112 | |
20 | |
| 113 | |
21 | |
| 12 | Freeze- |
22 | |
| 13 | |
30 | |
| 14 | |
40 | |
| 15 | Sealing film |
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all directional indicators (such as up, down, left, right, front, and back \8230;) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the motion situation, etc. in a specific posture (as shown in the attached drawings), and if the specific posture is changed, the directional indicators are changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise explicitly stated or limited, the terms "connected", "fixed", and the like are to be understood broadly, for example, "fixed" may be fixedly connected, may be detachably connected, or may be integrated; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.
The description of the orientations of "up", "down", "front", "back", "left", "right", etc. in the present invention is based on the orientation shown in fig. 1 and is only used to explain the relative positional relationship between the components in the orientation shown in fig. 1, and if the specific orientation is changed, the directional indication is changed accordingly.
The present invention provides a nucleic acid analysis card 100.
In one embodiment, as shown in fig. 1 to 5, the nucleic acid analysis card 100 includes a main body 10, a cover 20 and a phase-change material 30, the main body 10 forms a detection channel 11, the detection channel 11 includes a reagent region 111, a phase-change region 112 and a reaction region 113 which are sequentially connected, a lyophilized pellet 12 is disposed in the reagent region 111, the lyophilized pellet 12 includes an amplification detection reagent, the main body 10 is provided with a sample port 13, and the sample port 13 is connected to the reagent region 111; the buckle cover 20 is used for opening or closing the sample adding port 13; the phase change material 30 is disposed in the phase change region 112, and the phase change material 30 forms a liquid seal after being heated and melted.
The detection channel 11 includes a reagent zone 111, a phase change zone 112, and a reaction zone 113, which are sequentially connected from top to bottom. The cover 20 is used to open or close the sample inlet 13, the cover 20 opens the sample inlet 13, and the sample to be tested enters the reagent region 111 from the sample inlet 13. The lid 20 closes the sample addition port 13 to close the detection channel 11 for sufficient reaction, thereby preventing the reaction solution from leaking out of the sample addition port 13. The freeze-drying ball 12 is arranged in the reagent zone 111, the freeze-drying ball 12 comprises an amplification detection reagent, and compared with drying, freeze-drying has higher redissolution and mixing efficiency, and is suitable for full-component freeze-drying. The reagent area 111 is used for pre-storing and redissolving the lyophilized spheres 12, after the sample to be tested is added into the reagent area 111, the nucleic acid analysis card 100 is shaken to redissolve the nucleic acid analysis lyophilized spheres 12 of all components to form reaction liquid, and then the reaction liquid enters the reaction area 113 through the phase change area 112. The phase change material 30 forms a liquid seal after being heated and melted to seal the reaction area 113, and compared with a solid paraffin valve, the sealing is more reliable, and the liquid seal is re-solidified along with the temperature reduction after the reaction is finished to seal the reaction liquid, so that the reliability of the test result is improved.
According to the invention, the phase-change material 30 is arranged in the phase-change area 112, the phase-change material 30 is heated and melted to form a liquid seal to seal the reaction area 113, the sealing is more reliable compared with the sealing of a paraffin valve in a solid state, and the liquid seal is re-solidified along with the temperature reduction after the reaction is finished to seal the reaction liquid, so that the loss of the reaction liquid and the cross pollution with the environment, especially the aerosol pollution, are avoided, and the reliability of the test result is improved.
The main body 10 and the cover 20 are made of one or a combination of polypropylene, polycarbonate, polymethyl methacrylate and polystyrene, and have chemical resistance, heat resistance, electrical insulation, high-strength mechanical properties, good high-wear-resistance processability and the like, so that the nucleic acid analysis card 100 has a more reliable structure and better sealing performance, and avoids leakage of reaction liquid.
The main body 10 and the buckle cover 20 are formed by one or a combination of injection molding, machining, 3D printing and the like using the above materials. The production speed is high, the efficiency is high, the product size is accurate, and the product is convenient to update.
The minimum aperture range of the sample inlet 13 is 3mm to 7mm, and the maximum aperture range is 3.2mm to 7.5mm. The volume of the reagent zone 111 is 30 to 200 microliters, the diameter range of the freeze-drying ball 12 is 1mm to 5mm, the diameter of the freeze-drying ball 12 which is easy to stably produce in mass is often more than 2mm, the freeze-drying powder is difficult to be uniformly subpackaged, and the problem of extremely high cost exists when in-situ freeze-drying is carried out based on the shape of the reaction zone 113.
The amplification detection reagent is one of PCR, RT-PCR, LAMP, RT-LAMP, RPA, RT-RPA, TMA, NASBA, HAD, NEAR, RCA, SDA, ERA, RAA and MIRA, so as to improve the detection effect and reliability.
The amplification detection reagent comprises a dye, wherein the dye is one of a self-luminous fluorescent dye, a chimeric fluorescent dye and a pH indicator so as to facilitate the observation of a detection result.
The self-luminous fluorescent dye is one of hydroxytheaphenol blue (HNB) and Calcein (Calcein), the mosaic fluorescent dye is one of SYBR Green I, eva Green and Syto 82, and the pH indicator is one of phenol red (phenol red), cresol red (cresolred), neutral red (neutral red) and cresol purple (cresol purple) so as to observe a detection result.
When the self-luminous fluorescent dye is used, the nucleic acid analysis can be visually detected by directly observing the color change of the reaction solution with naked eyes, and can be automatically completed by analyzing the change of the optical signal of the reaction region 113 through the optical detection module. The volume of the phase change region 112 is 8 microliters to 50 microliters, and the minimum distance between the sample addition port 13 and the reaction region 113 and the sealing membrane 15 is 0.3 millimeters to 0.7 millimeters.
The phase change region 112 is pre-stored with the phase change material 30 which is solid at normal temperature, and the volume of the phase change material 30 is 3 microliters to 30 microliters, so as to improve the reliability of the test result.
The pre-storing manner of the phase change material 30 is as follows: cooling and solidification remain in the phase change region 112 after dispensing in the solid state, or dripping and cooling and solidification remain in the phase change region 112 in the hot melt liquid state.
In an embodiment, referring to fig. 1 to 3 in combination, the cover 20 includes a cover 21 and a skirt 22 connected to each other, the skirt 22 extends along a radial direction of the cover 21, the cover 21 is used for opening or closing the sample addition port 13, and the skirt 22 is used for abutting against the main body 10.
The cover 20 can open or close the sample addition port 13 by arranging the cover 21, the cover 21 extends into the sample addition port 13 to close the sample addition port 13, and the cover 21 is separated from the sample addition port 13 to open the sample addition port 13. Lid 21's edge is connected with shirt rim 22, and shirt rim 22 is along lid 21's radial extension, shirt rim 22 be used for with main part 10 butt, carry out backstop and spacing through setting up shirt rim 22, avoid lid 21 excessively to stretch into sample port 13 and lead to lid 21 to fall into reagent district 111 and can't take out, simultaneously, also avoid rocking when nucleic acid analysis card 100 lid 21 breaks away from main part 10 and leads to sample port 13 to open and the reaction liquid spills.
In an embodiment, referring to fig. 1 to 3, the main body 10 includes a sample adding portion 16, the sample adding portion 16 is opened with a sample adding port 13, the skirt 22 is used for abutting against the sample adding portion 16, and the sample adding portion 16 protrudes in a direction away from the detection channel 11 so that an installation gap 17 is formed between the skirt 22 and the sample adding portion 16.
The main body 10 is provided with a sample addition part 16 to open the sample addition port 13 and abut against the skirt 22. The direction of keeping away from detection channel 11 of sample addition part 16 is protruding for be formed with installation clearance 17 between shirt rim 22 and the sample addition part 16, through setting up installation clearance 17, make things convenient for operating personnel to grasp shirt rim 22 so that buckle closure 20 breaks away from sample addition port 13, thereby realize opening sample addition port 13 and carry out the application of sample, make the use of nucleic acid analysis card 100 more convenient.
In one embodiment, referring to fig. 1 to 3 in combination, the main body 10 is formed with a stop surface 18, the stop surface 18 is disposed obliquely, and the stop surface 18 is used for stopping the freeze-dried ball 12 from entering the phase change region 112.
The stop surface 18 inclines from top to bottom towards the direction close to the detection channel 11, the stop surface 18 is used for stopping the freeze-dried ball 12, the volume of the reagent area 111 is larger than that of the phase change area 112 and the reaction area 113, the freeze-dried ball 12 is prevented from entering the phase change area 112 and the reaction area 113 to cause unreliable redissolution of the freeze-dried ball 12 before redissolution through the stop surface 18, unreliable redissolution is that the freeze-dried ball 12 is not in full contact with a sample to be detected to cause part or the whole freeze-dried ball 12 to maintain a solid form and cannot effectively participate in subsequent reaction, and the freeze-dried ball 12 can be redissolved reliably in the reagent area 111 through the stop surface 18.
In an embodiment, referring to fig. 1 to 3, a mounting groove 19 is formed in the main body 10, and the phase change material 30 is disposed in the mounting groove 19.
The mounting groove 19 is used for mounting the phase change material 30, and the pre-storage mode of the phase change material 30 is as follows: cooling and solidification remain in the phase change region 112 after dispensing in the solid state, or dripping and cooling and solidification remain in the phase change region 112 in the hot melt liquid state. In addition, the mounting groove 19 has a limiting effect on the phase change material 30, the phase change material 30 is retained in the mounting groove 19 in a solid state, and the wall of the mounting groove 19 limits the phase change material 30, so that the phase change material 30 is prevented from shaking.
In an embodiment, referring to fig. 1 to 3, the main body 10 includes a casing 14 and a sealing film 15 connected to each other, the sealing film 15 cooperates with the casing 14 to form the detection channel 11, the casing 14 is provided with a sample injection port 13, the sealing film 15 is used for connecting to the heating module, and the thickness of the sealing film 15 is 0.05mm to 0.2mm.
The sealing film 15 is one of a single-sided adhesive film, a pressure-sensitive film and a heat-sealing film, has a thickness of 0.05mm to 0.2mm, and is sealed with the shell 14 in a cold pressing or hot pressing mode.
When a heat-sealing film is used as the sealing film 15, the material of the sealing film 15 is the same as that of the body 10.
When the heat-sealing film is used as the sealing film 15, the heat-sealing film needs to be kept in an upward direction until the melted phase-change material is cooled and solidified again in the process of sealing the case 14 and the heat-sealing film.
In an embodiment, referring to fig. 1 to fig. 3, the phase change material 30 is one or a combination of solid paraffin nos. 48, 50, 52, 54, 56, 58, 60, 62, and 64, and the solid paraffin is pharmaceutical grade fully refined paraffin.
The solid paraffin is divided into different varieties according to the melting point, generally every 2 ℃, such as No. 52, no. 54, no. 56, no. 58 and other brands, the phase-change material 30 adopts the solid paraffin with the melting point slightly lower than the reaction starting temperature, and the solid paraffin has stable property and is not easy to generate chemical reaction with an amplification detection reagent and a sample to be detected. The solid paraffin is medical grade fully refined paraffin, and the high-purity solid paraffin is used, so that the liquid sealing effect and reliability are improved.
In an embodiment, referring to fig. 1 to fig. 3, the phase change material 30 is one or a combination of heneicosane, docosane, tricosane, tetracosane, pentacosane, hexacosane, heptacosane, octacosane, nonacosane and triacontane, and the purity of the phase change material 30 is greater than or equal to 95%.
The chemical property of the alkane is stable, and the alkane is not easy to generate chemical reaction with the amplification detection reagent and a sample to be detected. And the purity of the alkane is more than or equal to 95 percent, and the alkane with high purity is used, so that reliable liquid seal in the reaction process and effective seal after the reaction are finished are realized while the reaction result is not interfered.
Specifically, the phase change material 30 is one or a combination of heneicosane, docosane, tricosane and tetracosane, and the purity of the phase change material 30 is greater than or equal to 99%. Further limit the purity of the alkane and improve the liquid seal effect.
In an embodiment, referring to fig. 4, the number of the detection channels 11 is plural, the detection channels 11 are arranged at intervals, the number of the covers 20 and the number of the phase change materials 30 are plural, the number of the covers 20 is the same as that of the detection channels 11, and the number of the phase change materials 30 is the same as that of the detection channels 11.
A plurality of detection channel 11 set up along left right direction interval, buckle closure 20 and detection channel 11's quantity unanimity and one-to-one setting, buckle closure 20 is used for opening or closing sample addition mouth 13. The phase-change materials 30 and the detection channels 11 are in the same number and are arranged in a one-to-one correspondence manner, and the phase-change materials 30 are heated and melted to form a liquid seal. The number of the detection channels 11 is multiple, so that the simultaneous detection of multiple samples to be detected can be realized, and the detection efficiency is improved.
In one embodiment, referring to FIGS. 1 to 3, a handle 40 is connected to an end of the main body 10 near the reagent zone 111 to facilitate inserting the nucleic acid analysis card 100 downward into the heating module and also to facilitate pulling the nucleic acid analysis card 100 upward.
In addition, referring to fig. 5, the present invention further provides a method for using the nucleic acid analysis card 100, wherein the nucleic acid analysis card 100 includes the nucleic acid analysis card 100 as described above, and the method includes:
step S10, dripping a sample to be detected into the sample adding port 13;
the cover 20 is separated from the sample port 13 to open the sample port 13, a pipette, a quantitative pipette or other container is used to suck a sample to be tested with a fixed volume, the sample to be tested is dripped into the sample port 13, and the cover 20 is clamped into the sample port 13 to close the sample port 13.
Step S20, adjusting the direction of the nucleic acid analysis card 100 to the reagent zone 111 facing downwards, and shaking the nucleic acid analysis card 100 to redissolve the lyophilized pellet 12 to form a reaction solution;
the volume of the reagent area 111 is larger than that of the phase change area 112 and the reaction area 113, the reagent area 111 of the nucleic acid analysis card 100 faces downwards, and the nucleic acid analysis card 100 is shaken to enable the lyophilized ball 12 to be reliably redissolved in the reagent area 111, so that the phenomenon that the lyophilized ball 12 enters the phase change area 112 and the reaction area 113 to cause unreliable redissolution of the lyophilized ball 12 is avoided, wherein the unreliable redissolution is that the lyophilized ball 12 does not fully contact with a sample to be detected to cause part or the whole lyophilized ball 12 to maintain a solid state and cannot effectively participate in subsequent reactions.
Step S30, adjusting the direction of the nucleic acid analysis card 100 until the reagent zone 111 faces upwards, and swinging the nucleic acid analysis card 100 to make all the reaction solution fall into the reaction zone 113;
all the reaction liquid falls into the reaction area 113, which facilitates the operator to observe the optical signal in the reaction area 113 and facilitates the phase change material 30 to form a liquid seal.
Step S40, heating the nucleic acid analysis card 100 to melt the phase change material 30 and flow the phase change material to the upper layer of the reagent in the reaction region 113 to form a liquid seal;
heating is carried out through the heating module for phase change material 30 melts, becomes liquid state from the solid state, then phase change material 30 forms the liquid seal because of the upper strata of reaction liquid in gravity and the density Liudong will reaction zone 113 to seal reaction zone 113, it is more reliable to compare in the paraffin valve seal of solid form.
S50, standing for a preset time within a preset temperature range, and finishing amplification of the reaction liquid within the preset time;
in the preset temperature range, the preset temperature range can be flexibly adjusted according to the actual reaction requirement, and the preset temperature range is not limited by the invention. The liquid seal makes the nucleic acid analysis card 100 suitable for isothermal amplification and variable-temperature amplification, and the paraffin wax valve is only suitable for isothermal amplification, so the use of the liquid seal also makes the nucleic acid analysis card 100 more convenient.
And standing for a preset time length, wherein the preset time length is flexibly adjusted according to actual reaction requirements, and the preset time length is not limited by the method. The reaction solution completes amplification within a preset time.
Step S60, stopping heating when the standing time reaches the preset time;
when the standing time reaches the preset time, the heating is stopped, and the phase-change material 30 is re-solidified along with the temperature reduction to seal the reaction liquid, so that the reaction liquid stays in the reaction area 113, and an operator can observe the optical signal in the reaction area 113 conveniently.
Step S70, when the phase change material is re-solidified, a nucleic acid analysis result is obtained according to the change of the optical signal in the reaction region 113.
The specific structure of the nucleic acid analysis card 100 refers to the above embodiments, and since the using method adopts all technical solutions of the above embodiments, at least all the beneficial effects brought by the technical solutions of the above embodiments are achieved, and no further description is given here.
Example 1 Single-channel nucleic acid analysis card 100
The parameters in this embodiment are as follows: the nucleic acid analysis card 100 includes a body 10 and a cover 20, the body 10 including a case 14 and a sealing film 15; the minimum aperture and the maximum aperture of the sample addition port 13 are respectively 5.2mm and 5.4mm, and the minimum distance between the sample addition port 13 and the sealing membrane 15 is 5mm; the volume of the reagent zone 111 is 75 μ l, and the diameter of the freeze-dried ball 12 is 4mm; the volume of the phase change region 112 is 30 μ l, and the minimum distance from the sealing film 15 is 0.6mm; the phase change material 30 is No. 52 medical paraffin, and the volume is 16 mu l; the reaction zone 113 had a volume of 60. Mu.l, a distance of 1mm from the bottom surface of the sealing film 15, and a thickness of 1mm at the bottom surface; the body 10 and the cover 20 are made of white PP material, and the seal 3 is made of a transparent PP heat sealing film with a thickness of 0.08 mm.
The preparation of the single-channel nucleic acid analysis card 100 based on the above parameters includes the steps of:
1) Performing injection molding on the main body 10 and the buckle cover 20, and performing cleaning and autoclaving;
2) Preparing paraffin particles and subpackaging the paraffin particles into the phase change area 112 of the main body 10;
3) The whole body 10 is preheated to 70 ℃, paraffin is completely liquefied, and then a heat sealing film is sealed on the body 10 by using a film hot-pressing device and is conveyed to a cooling area;
4) After the paraffin is cooled and solidified in the phase change region 112, the lyophilized pellet 12 is dispensed to the reagent region 111 through the sample port 13 in a clean environment with a humidity of 25%, and finally the cover 20 is fastened back to close the sample port 13 for storage at normal or low temperature.
Example 2 detection of New coronavirus, influenza Virus and respiratory syncytial Virus in a sample Using a 4-channel nucleic acid analysis card 100 with RT-LAMP & HNB reagents
The parameters in this embodiment are as follows: the nucleic acid analysis card 100 includes a body 10 and a cover 20, the body 10 including a case 14 and a sealing film 15; the main body 10 comprises 4 sample ports 13 and 4 detection channels 11, and the distance between the central lines of the two adjacent detection channels 11 is 9mm; the minimum aperture and the maximum aperture of the sample addition port 13 are respectively 5.2mm and 5.6mm, and the minimum distance between the sample addition port 13 and the sealing membrane 15 is 0.6mm; the volume of the reagent zone 111 is 75 μ l, and the diameter of the freeze-dried ball 12 is 4mm; the volume of the phase change region 112 is 30 μ l, and the minimum distance of the sealing film 15 is 0.6mm; the phase change material 30 is No. 52 medical paraffin, and the volume is 16 mu l; the volume of the reaction zone 113 is 60 μ l, the distance from the bottom surface to the sealing film 15 is 1mm, and the thickness of the bottom surface is 0.8mm; the body 10 and the cap 20 are made of white PP, and the sealing film 15 is made of a transparent PP heat sealing film having a thickness of 0.08 mm.
The reagent composition in the freeze-dried ball 12 comprises a primer, HNB, dNTP, BST DNA/RNA polymerase, magnesium chloride and other buffer reagents, wherein primer probes in four detection channels 11 respectively correspond to a human cell Actin gene, a new coronavirus N gene, an influenza virus M gene and a respiratory syncytial virus F gene.
The assay card was prepared according to the same procedure as in example 1.
The method for detecting the new coronavirus, the influenza virus and the respiratory syncytial virus of the sample to be detected based on the parameters comprises the following steps:
1) Treating the sampled throat swab with the sample releasing agent, and opening the cover 20 to open the sample addition port 13;
2) Sucking a sample to be detected by using a 50-microliter quantitative pipette, adding the sample into the reagent area 111 through the sample adding opening 13, repeatedly adding the sample for 4 times to finish the sample adding of 4 detection channels 11, and then buckling the buckling cover 20 back to close the sample adding opening 13;
3) Inverting the nucleic acid analysis card 100 to make the reagent zone 111 face downwards, shaking the nucleic acid analysis card 100 to promote the re-dissolution of the freeze-dried reagent, inverting the nucleic acid analysis card 100 to make the reagent zone 111 face upwards, shaking the nucleic acid analysis card 100 to make all reagents fall to the bottom of the reaction zone 113, wherein the color of the reaction solution is light purple;
4) Inserting the nucleic acid analysis card 100 into a 65 ℃ heating module, counting down for 30 minutes, and taking out;
5) Visually observing the color change of each reaction region 113, for example, from left to right, the colors are sky blue, light purple, sky blue, and light purple, and the corresponding detection results are: the sample collection and reaction are effective, positive influenza virus is detected, and new coronavirus and respiratory syncytial virus are not detected.
Example 3 detection of neocoronavirus, influenza virus and respiratory syncytial virus in a sample Using a 4-channel nucleic acid analysis card 100 with RT-LAMP & SYBR Green I reagent
The parameters in this embodiment are as follows: the nucleic acid analysis card 100 includes a body 10 and a cover 20, the body 10 including a case 14 and a sealing film 15; the main body 10 comprises 4 sample adding ports 13 and 4 detection channels 11, and the distance between the central lines of two adjacent detection channels 11 is 9mm; the minimum aperture and the maximum aperture of the sample adding port 13 are respectively 5.2mm and 5.6mm, and the minimum distance between the sample adding port and the sealing membrane 15 is 0.6mm; the volume of the reagent zone 111 is 75 mul, and the diameter of the freeze-drying ball 12 is 4mm; the volume of the phase change region 112 is 30 μ l, and the minimum distance to the sealing film 15 is 0.6mm; the phase change material 30 is n-tetracosane with 99% purity and the volume is 16 mu l; the reaction zone 113 had a volume of 60. Mu.l, a distance of 1mm from the bottom surface of the sealing film 15, and a thickness of 0.8mm at the bottom surface; the body 10 and the button cover 20 are made of a transparent PP material, and the sealing film 15 is a transparent PP heat sealing film having a thickness of 0.08 mm.
The reagent composition in the freeze-dried ball 12 comprises primers, SYBR Green I, dNTP, BST DNA/RNA polymerase, magnesium chloride and other buffer reagents, wherein the primer probes in the four detection channels 11 respectively correspond to a human cell Actin gene, a new coronavirus N gene, an influenza virus M gene and a respiratory syncytial virus F gene.
The assay card was prepared according to the same procedure as in example 1.
The method for detecting the new coronavirus, the influenza virus and the respiratory syncytial virus of the sample to be detected based on the parameters comprises the following steps:
1) Treating the sampled throat swab with the sample releasing agent, and opening the cover 20 to open the sample addition port 13;
2) Sucking up a sample by using a 50 mu l quantitative pipette, adding the sample into the reagent area 111 through the sample adding port 13, repeatedly adding the sample for 4 times to finish the sample adding of 4 channels, and then buckling the buckling cover 20 back to close the sample adding port 13;
3) Inverting the nucleic acid analysis card 100 to make the reagent zone 111 face downwards, shaking the nucleic acid analysis card 100 to promote the re-dissolution of the freeze-dried reagent, inverting the nucleic acid analysis card 100 to make the reagent zone 111 face upwards, and shaking the nucleic acid analysis card 100 to make all the reagents fall to the bottom of the reaction zone 113;
4) Inserting the nucleic acid analysis card 100 into a 65 ℃ heating module, and taking out after counting for 30 minutes;
5) The reaction regions 113 of the nucleic acid analysis card 100 are irradiated by an ultraviolet light source, and whether green fluorescence appears in each reaction region 113 is observed, the colors from left to right are colorless, green, colorless, and the corresponding detection results are as follows: the sample collection and reaction are effective, the new coronavirus positive is detected, and the influenza virus and the respiratory syncytial virus are not detected.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A nucleic acid analysis card, comprising:
the kit comprises a main body, a detection channel and a detection device, wherein the main body is provided with the detection channel, the detection channel comprises a reagent area, a phase change area and a reaction area which are sequentially communicated, a freeze-dried ball is arranged in the reagent area, the freeze-dried ball comprises an amplification detection reagent, the main body is provided with a sample adding port, and the sample adding port is communicated with the reagent area;
the buckle cover is used for opening or closing the sample adding port;
the phase-change material is arranged in the phase-change area and forms a liquid seal after being heated and melted.
2. The nucleic acid analysis card according to claim 1, wherein the retaining cap comprises a cap body and a skirt connected to each other, the skirt extending radially of the cap body, the cap body being adapted to open or close the sample addition port, and the skirt being adapted to abut against the main body.
3. The nucleic acid analysis card of claim 2, wherein the main body comprises a sample addition part, the sample addition part is provided with the sample addition port, the skirt is used for abutting against the sample addition part, and the sample addition part protrudes in a direction away from the detection channel so that an installation gap is formed between the skirt and the sample addition part.
4. The nucleic acid analysis card of claim 1, wherein the body is formed with a stop surface, the stop surface being disposed obliquely, the stop surface being for stopping the lyophilized pellet from entering the phase transition region.
5. The nucleic acid analysis card of claim 1, wherein the main body is formed with a mounting groove, and the phase change material is disposed in the mounting groove.
6. The nucleic acid analysis card of claim 1, wherein the main body comprises a housing and a sealing membrane connected with each other, the sealing membrane and the housing cooperate to form the detection channel, the housing is provided with the sample addition port, the sealing membrane is used for being connected with a heating module, and the thickness of the sealing membrane is 0.05mm to 0.2mm.
7. The nucleic acid analysis card of any one of claims 1 to 6, wherein the phase change material is one or a combination of 48, 50, 52, 54, 56, 58, 60, 62 and 64 paraffin wax, and the paraffin wax is pharmaceutical grade fully refined paraffin wax.
8. The nucleic acid analysis card of any one of claims 1 to 6, wherein the phase change material is one or a combination of heneicosane, docosane, tricosane, tetracosane, pentacosane, hexacosane, heptacosane, octacosane, nonacosane, triacontane, and the phase change material has a purity of 95% or more.
9. The nucleic acid analysis card according to any one of claims 1 to 6, wherein the number of the detection channels is plural, the plurality of detection channels are arranged at intervals, the number of the caps and the number of the phase change materials are plural, the caps and the detection channels are in one-to-one correspondence, and the phase change materials and the detection channels are in one-to-one correspondence.
10. A method of using a nucleic acid analysis card comprising the nucleic acid analysis card of any one of claims 1 to 9, the method comprising:
dripping a sample to be detected into the sample adding port;
adjusting the direction of the nucleic acid analysis card to the downward direction of the reagent area, and shaking the nucleic acid analysis card to redissolve the freeze-dried balls to form a reaction solution;
adjusting the direction of the nucleic acid analysis card until the reagent area is upward, and swinging the nucleic acid analysis card to make all reaction liquid fall into the reaction area;
heating the nucleic acid analysis card to melt the phase-change material and make the phase-change material flow to the upper layer of the reaction solution in the reaction area to form a liquid seal;
standing for a preset time within a preset temperature range, and finishing amplification of the reaction liquid within the preset time;
when the standing time reaches the preset time, stopping heating;
when the phase-change material is re-solidified, a nucleic acid analysis result is obtained according to the change of the optical signal in the reaction area.
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Application publication date: 20230307 |