CN114317238A - Nucleic acid detecting cassette and nucleic acid detecting apparatus - Google Patents
Nucleic acid detecting cassette and nucleic acid detecting apparatus Download PDFInfo
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- CN114317238A CN114317238A CN202110735245.9A CN202110735245A CN114317238A CN 114317238 A CN114317238 A CN 114317238A CN 202110735245 A CN202110735245 A CN 202110735245A CN 114317238 A CN114317238 A CN 114317238A
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Abstract
A nucleic acid detection box and nucleic acid detection equipment, the nucleic acid detection box includes box body, detection chip and electrophoresis box, the detection chip locates in box body, the detection chip includes the first cover plate, spacer layer and second cover plate, two surfaces opposite to spacer layer border on first cover plate and second cover plate separately, the first cover plate, spacer layer and second cover plate enclose and form the channel, the channel is used for bearing the detection liquid in order to make the detection liquid carry on the nucleic acid amplification reaction in the channel and thus get the nucleic acid amplification product; the electrophoresis box is arranged outside the box body and communicated with the channel, and the electrophoresis box is used for carrying out electrophoresis detection on the nucleic acid amplification product. The nucleic acid detection kit provided by the invention integrates nucleic acid amplification reaction and electrophoresis detection, and has the advantages of simple and convenient detection operation, high efficiency, low cost and strong detection flexibility.
Description
Technical Field
The present invention relates to a nucleic acid detecting cassette and a nucleic acid detecting apparatus.
Background
Currently, most of the tests for molecular diagnosis, morphology, immunology, etc. are performed in professional laboratories, and the conventional testing process generally includes the following steps: firstly, carrying out nucleic acid amplification by large and medium-sized detection equipment; then, manually transferring the amplified nucleic acid to electrophoresis detection equipment for electrophoresis detection; and finally, manually transferring the electrophoresis detection result to a special fluorescence analyzer for result analysis. The whole detection process requires complicated equipment, large volume, low detection efficiency, poor flexibility, high cost, complex operation and higher requirement on the professional level of operators, and the detection process needs to be operated by skilled technicians and cannot realize household portable detection.
Disclosure of Invention
In view of the above, in order to overcome at least one of the above-mentioned drawbacks, it is necessary to provide a nucleic acid detecting cassette.
In addition, the application also provides a nucleic acid detection device.
The invention provides a nucleic acid detection box, which comprises a box body, a detection chip and an electrophoresis box, wherein the detection chip is arranged in the box body and comprises a first cover plate, a spacing layer and a second cover plate, two opposite surfaces of the spacing layer are respectively adjacent to the first cover plate and the second cover plate, the first cover plate, the spacing layer and the second cover plate are surrounded to form a channel, and the channel is used for bearing detection liquid so that the detection liquid can carry out nucleic acid amplification reaction in the channel to obtain a nucleic acid amplification product; the electrophoresis box is arranged outside the box body and is communicated with the channel, and the electrophoresis box is used for carrying out electrophoresis detection on the nucleic acid amplification product.
In the embodiment of the application, the detection chip further comprises a driving circuit arranged on one side of the second cover plate and a driving circuit arranged on one side of the second cover plate, wherein the second cover plate is arranged on one side of the first cover plate, a conducting layer arranged on one side of the first cover plate and a first dielectric layer arranged on one side of the second cover plate, and the conducting layer is arranged on one side of the second cover plate and a second dielectric layer arranged on one side of the first cover plate, the driving circuit is electrically connected with the conducting layer through external electrophoresis, and the channel is formed between the first dielectric layer and the second dielectric layer.
In the embodiment of the present application, the driving circuit includes at least one nucleic acid amplification region, the cartridge body is provided with at least one heating opening corresponding to the nucleic acid amplification region, and the detection chip is exposed from the heating opening.
In the embodiment of the application, the box body comprises an upper shell and a lower shell, wherein the upper shell and the lower shell correspond to the nucleic acid amplification zone and are respectively provided with the heating port.
In the embodiment of the application, drive circuit includes a plurality of drive electrodes that are the array and arrange, drive electrode is connected with external power supply, control drive electrode with circular telegram or outage between the conducting layer, so that detect liquid adjacent two move between the drive electrode.
In the embodiment of the application, still include with each the driving electrode with conducting layer electric connection's control panel, the control panel is located first apron is close to a side surface of second apron, just the control panel is located the outside of passageway, the box body corresponds the control panel is equipped with the connector, the control panel by the connector exposes.
In the embodiment of the application, the driving circuit further comprises a sample adding area, a reagent storage area and a liquid outlet area, and the liquid outlet area is communicated with the electrophoresis box.
In the embodiment of the application, a reagent bag is arranged in the reagent storage area, a fluorescent reagent is contained in the reagent bag, the reagent bag extends out of the first cover plate, a reagent groove is formed in the box body corresponding to the reagent storage area, and the reagent bag is contained in the reagent groove.
In this application embodiment, the electrophoresis box includes that electrophoresis tank, lid close electrophoresis tank apron on the electrophoresis tank, locate respectively two electrophoresis electrodes at the inside both ends of electrophoresis tank, locate two gel medium between the electrophoresis electrode and locating the notes liquid groove of gel medium one end, each electrophoresis electrode all with external power electric connection, first apron corresponds the notes liquid groove is equipped with the liquid outlet, the liquid outlet with annotate liquid groove intercommunication.
In the embodiment of the application, be equipped with first porous adsorption block in the notes liquid groove, bear the weight of wetting fluid in the first porous adsorption block, first porous adsorption block one end stretches into the bottom in notes liquid groove, other end parallel and level or protrusion the liquid outlet is close to the surface of passageway.
In the embodiment of the application, be equipped with the porous adsorption block of second in the notes liquid groove, the weight of dampening solution bears in the porous adsorption block of second, liquid outlet department is equipped with the adsorption tube, adsorption tube one end is inserted in the porous adsorption block of second, other end parallel and level or protrusion the liquid outlet is close to the surface of passageway, in the porous adsorption block of second dampening solution is full of the adsorption tube.
The present invention also includes a nucleic acid detecting apparatus including: the nucleic acid detection device comprises a host and a detection box mounting area, wherein the detection box mounting area is arranged on the host, the detection box mounting area is used for detachably mounting a nucleic acid detection box, and the nucleic acid detection box is as above.
In the embodiment of the application, the host comprises a host connector, a heating device and an image acquisition device, the host connector is arranged in the detection box mounting area, and the host is connected with the machine and is used for electrically connecting the detection chip and the electrophoresis box; the heating device is arranged in the detection box mounting area and used for heating the detection chip; the image acquisition device is arranged in the detection box mounting area and corresponds to the electrophoresis box, and the image acquisition device is used for acquiring images of the electrophoresis box.
Compared with the prior art, the nucleic acid detection kit provided by the invention integrates nucleic acid amplification reaction and electrophoresis detection, has a simple overall structure, is simple and convenient to detect and operate, has low professional requirements on the operation process, is high in detection efficiency, and greatly reduces the cost; meanwhile, the flexibility of the detection process is strong, the detection process does not need to be carried out in a fixed laboratory, and the nucleic acid detection box is portable and can realize community detection or family detection.
Drawings
FIG. 1 is a schematic front view of a nucleic acid detecting cassette according to an embodiment of the present invention.
FIG. 2 is a schematic view showing a back side structure of the nucleic acid detecting cassette according to the embodiment of the present invention.
FIG. 3 is an exploded view of a nucleic acid detecting cassette according to an embodiment of the present invention.
FIG. 4 is an exploded view of a part of a nucleic acid detecting cassette according to an embodiment of the present invention.
Fig. 5 is a schematic structural diagram of a detection chip according to an embodiment of the present invention.
Fig. 6 is a schematic cross-sectional view of a detection chip according to an embodiment of the invention.
Fig. 7 is a schematic structural diagram of a TFT driving circuit in a detection chip according to an embodiment of the present invention.
Fig. 8 is a schematic structural diagram of an electrophoresis cassette according to an embodiment of the present invention.
Fig. 9 is a schematic structural view of an electrophoresis cassette provided in another embodiment of the present invention, which is in communication with a channel.
Fig. 10 is a schematic structural view of an electrophoresis cassette provided in an embodiment of the present invention and communicating with a channel.
FIG. 11 is a schematic structural view of a nucleic acid detecting apparatus according to an embodiment of the present invention.
FIG. 12 is a partial sectional view of a nucleic acid detecting apparatus according to an embodiment of the present invention.
FIG. 13 is a diagram showing the result of the detection by electrophoresis in a nucleic acid according to an embodiment of the present invention.
Description of the main elements
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
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.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When a component is referred to as being "connected" to another component, it can be directly connected to the other component or intervening components may also be present. When a component is referred to as being "disposed on" another component, it can be directly on the other component or intervening components may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only.
The system embodiments described below are merely illustrative, and the division of the modules or circuits is merely a logical division, and other divisions may be realized in practice. Furthermore, it is obvious that the word "comprising" does not exclude other elements or steps, and the singular does not exclude the plural. Several units or means recited in the system claims may also be implemented by one and the same unit or means in software or hardware. The terms first, second, etc. are used to denote names, but not any particular order.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to fig. 1 to 7, a nucleic acid detecting cassette 100 according to an embodiment of the invention includes a cassette body 1, a detecting chip 2 and an electrophoresis cassette 3. The detection chip 2 is arranged in the box body 1, and the electrophoresis box 3 is arranged outside the box body 1. The detection chip 2 includes a first cover plate 21, a spacer layer 22 and a second cover plate 23, two opposite surfaces of the spacer layer 22 are respectively adjacent to the first cover plate 21 and the second cover plate 23, the first cover plate 21, the spacer layer 22 and the second cover plate 23 enclose a channel 5, and the channel 5 is used for carrying detection liquid a. The electrophoresis cassette 3 is in communication with the channel 5. The detection chip 2 and the electrophoresis box 3 are both electrically connected with an external power supply 7. The nucleic acid detecting cassette 100 is used for performing nucleic acid amplification reaction and electrophoresis detection, a detecting liquid a containing a nucleic acid sample is added into a channel 5 of the detecting chip 2, it should be noted that the detecting liquid a exists in the channel 5 in the form of liquid beads, the detecting liquid a performs nucleic acid amplification reaction in the channel 5 to obtain a nucleic acid amplification product (the nucleic acid amplification product is combined with a fluorescent reagent), the nucleic acid amplification product directly enters the electrophoresis cassette 3 from the detecting chip 2 for electrophoresis detection, and finally an image of the electrophoresis cassette 3 is captured by an image collecting device matched with the nucleic acid detecting cassette 100, wherein the image is a fluorescent photograph of the electrophoresis detection. According to the invention, the detection chip 2 and the electrophoresis box 3 are integrated, the whole structure is simple, complex large-scale equipment is not needed, the cost is low, the detection liquid a can directly enter the electrophoresis box 3 for electrophoresis detection after completing nucleic acid amplification, the sample transfer matching connection process of different detection links is simplified, and the detection efficiency is improved.
Referring to fig. 6, the detecting chip 2 further includes a driving circuit 24 disposed on a side of the first cover plate 21 close to the second cover plate 23, a first dielectric layer 26 disposed on a side of the driving circuit 24 close to the second cover plate 23, a conductive layer 25 disposed on a side of the second cover plate 23 close to the first cover plate 21, and a second dielectric layer 27 disposed on a side of the conductive layer 25 close to the first cover plate 21, wherein the driving circuit 24 and the conductive layer 25 are both electrically connected to an external power source 7, and the detecting liquid a can move in the channel 5 according to a predetermined path by turning on or off the driving circuit 24 and the conductive layer 25 through the external power source 7.
In this embodiment, as shown in fig. 6 and 7, the driving circuit 24 includes a plurality of driving electrodes 241 arranged in an array, the nucleic acid detecting cassette 100 further includes a control board 4, the control board 4 is electrically connected to each of the driving electrodes 241 and the conductive layer 25, the control board 4 is disposed on a side surface of the first cover plate 21 close to the second cover plate 23, and the control board 4 is located outside the channel 5 and can be electrically connected to the external power source 7 through the control board 4.
In the present embodiment, the driving circuit 24 is a Thin Film Transistor (TFT) driving circuit, and since the detection liquid a has conductivity, the detection liquid a can move along a predetermined path in the channel 5 by combining an electro wetting-On-Dielectric (EWOD) principle. Using the TFT principle, a circuit between a certain driving electrode 241 and the conductive layer 25 can be selectively turned on or off, so that the voltage between the driving electrode 241 and the conductive layer 25 is changed to change the wetting characteristics between the detection liquid a and the first and second dielectric layers 26 and 27, thereby controlling the detection liquid a to move along a predetermined path in the channel 5. As shown in fig. 6, the detection liquid a moves on the electrode I, the electrode H and the electrode G, and when the detection liquid a is on the electrode H, a voltage is applied between the electrode G and the conductive layer 25, a voltage Vd is applied to the electrode G, and the voltage between the electrode H and the conductive layer 25 is disconnected, at which time the wetting characteristics between the detection liquid a and the first dielectric layer 26 and the second dielectric layer 27 are changed, so that the liquid-solid contact angle between the electrode H and the detection liquid a becomes larger, and the liquid-solid contact angle between the electrode G and the detection liquid a becomes smaller, thereby urging the detection liquid a to move from the electrode H to the electrode G.
In this embodiment, the first dielectric layer 26 and the second dielectric layer 27 are both insulating hydrophobic layers, and may be specifically polytetrafluoroethylene coatings, which can play a role of insulating and hydrophobic on the one hand, and can also make the detection liquid a move more smoothly in a specified path on the other hand, thereby avoiding the liquid bead from breaking during the moving process.
In the present embodiment, referring to fig. 7, the driving circuit 24 is disposed on a side of the first cover plate 21 close to the channel 5. The driving circuit 24 may be formed by a metal etching method or an electroplating method.
Referring to FIG. 7 in conjunction with FIG. 4, the driving circuit 24 can be divided into a plurality of regions, namely, a sample application region A, a reagent storage region B, at least one nucleic acid amplification region C and a liquid discharge region D, according to different applications. The sample addition part A is used for adding the detection liquid a. The reagent storage region B is used to store a fluorescent reagent (e.g., a fluorescent dye or a fluorescent probe). The detection solution a performs a nucleic acid amplification reaction in the nucleic acid amplification region C, which may include a plurality of regions, and the number of the specific regions may be determined according to the actual detection requirement. The liquid outlet area D is communicated with the electrophoresis box 3, and the nucleic acid amplification product combined with the fluorescent reagent can enter the electrophoresis box 3 through the liquid outlet area D for electrophoresis detection.
Referring to fig. 7 again, with reference to fig. 4 and fig. 6, the specific moving path of the detection liquid a in the detection chip 2 is: after entering the sample addition region A, the detection solution a moves to the nucleic acid amplification region C according to a predetermined path under the drive of the drive electrode 241 to perform an amplification reaction; when the amplification reaction is completed, the amplified product moves to the reagent storage area B to be mixed with the fluorescent reagent, so that a nucleic acid amplification product combined with the fluorescent reagent is obtained; the nucleic acid amplification product combined with the fluorescent reagent moves to the liquid outlet area D by the driving electrode 241 and enters the electrophoresis cassette 3 through the liquid outlet 51 of the liquid outlet area D.
It is understood that for more complete mixing, the nucleic acid amplification product can be moved back and forth in the nucleic acid amplification region C under the driving of the driving electrode 241, so that the amplification product and the fluorescent reagent are uniformly mixed. It is also understood that a mixing region may be separately provided to achieve sufficient mixing of the detection solution a and the fluorescent reagent in the nucleic acid amplification product.
In this embodiment, the number of the nucleic acid amplification regions C is two, and the heating temperatures of the two nucleic acid amplification regions C are different, so that the detection solution a can be subjected to different stages of nucleic acid amplification reactions at different temperatures.
In this embodiment, the specific heating temperature ranges of the two nucleic acid amplification regions C are 40 ℃ to 75 ℃ and 90 ℃ to 105 ℃, respectively.
In other embodiments, the number of nucleic acid amplification regions C may be three or more depending on the stage of a particular nucleic acid amplification reaction. Specifically, the heating temperatures corresponding to the nucleic acid amplification region C are in the ranges of 40 ℃ to 65 ℃, 68 ℃ to 75 ℃ and 90 ℃ to 105 ℃, respectively.
Referring to fig. 3 and 4 in combination, referring to fig. 7 in combination, in the present embodiment, a reagent bag 6 is disposed in the reagent storage region B, a fluorescent reagent is disposed in the reagent bag 6, the reagent bag 6 extends out of the second cover plate 23, the reagent storage region B of the box body 1 is provided with a reagent slot 16, the reagent bag 6 is accommodated in the reagent slot 16, and during the detection process, when the nucleic acid amplification product needs to be combined with the fluorescent reagent, the reagent slot 16 is heated by an external heating device, so that the reagent bag 6 is melted, and the fluorescent reagent enters the channel 5 to be combined with the amplified product.
In another embodiment, the fluorescent reagent is pre-coated in the reagent storage region B during the assembly of the detection chip 2, and there is no need to separately add the fluorescent reagent or dispose the reagent capsule 6.
In the present embodiment, after the detection chip 2 is assembled, silicone oil is injected into the channel 5 through the injection hole of the sample application region a, and the detection liquid a moves through a predetermined path in the silicone oil.
Referring to fig. 5 and 6, in the present embodiment, the first cover plate 21 and the second cover plate 23 are both glass plates, and the spacer layer 22 is a double-sided adhesive frame, and is adhered to the edges of the first cover plate 21 and the second cover plate 23 through the double-sided adhesive frame, so as to jointly form a sealed channel 5. Wherein the capacity of the channel 5 can be adjusted by designing spacer layers 22 with different thicknesses according to actual requirements.
Referring to fig. 1 to fig. 3, with reference to fig. 4, the cartridge 1 includes a first housing 11, a second housing 12, a sample port 13, a connection port 15, and a heating port 14, the first housing 11 and the second housing 12 together form a containing cavity (not shown), the detecting chip 2 is contained in the containing cavity, and the electrophoresis cartridge 3 is located on a side of the first housing 11 away from the second housing 12. The sample addition port 13 and the connection port 15 are provided in the second casing 12, and the heating port 14 is provided in the first casing 11 and/or the second casing 12. The sample addition port 13 is provided corresponding to the pressure region A of the detection chip 2, is communicated with the sample addition region A, and is used for adding a detection solution a containing a nucleic acid sample into the detection chip 2. The connection port 15 is provided corresponding to the control board 4, the control board 4 is exposed from the connection port 15, and the external power supply 7 can be inserted into the connection port 15 to be abutted against the control board 4 and electrically connected thereto. The heating port 14 is disposed corresponding to the nucleic acid amplification region C of the detection chip 2, the outer surface of the detection chip 2 corresponding to the nucleic acid amplification region C is exposed from the heating port 14, and an external heating device (not shown) extends into the heating port 14 to contact with the surface of the detection chip 2 to heat the nucleic acid amplification region C.
In this embodiment, the reagent well 16 is provided in the second casing 12, and the external heating device is provided on the side of the second casing 12 remote from the first casing 11 and in contact with the outer surface of the reagent well 16, so that when the nucleic acid amplification product needs to be mixed with the fluorescent reagent, the reagent bag 6 is melted by controlling the external heating device, and the fluorescent reagent in the reagent bag 6 is mixed with the nucleic acid amplification product.
In this embodiment, the heating ports 14 are formed in the first casing 11 and the second casing 12, and the external heating device can heat both the upper and lower surfaces of the detection chip 2 at the same time, so that the nucleic acid amplification region C is heated more uniformly, the heating efficiency is higher, and the nucleic acid amplification reaction is more sufficient. Specifically, the number of the heating ports 14 may be determined according to the number of the nucleic acid amplification regions C.
In this embodiment, there are two nucleic acid amplification regions C, and four heating ports 14.
Referring to fig. 3, in the present embodiment, the first housing 11 and the second housing 12 are bonded by an adhesive, and it can be understood that the first housing 11 and the second housing 12 can also be connected by a snap-fit manner, and are fastened by screws, so as to increase the connection firmness of the first housing 11 and the second housing 12.
Referring to fig. 1 and fig. 3, in the present embodiment, the box body 1 further includes two bumps 18, the two bumps 18 are disposed on two side walls of the box body 1, and the bumps 18 can facilitate the insertion and extraction of the nucleic acid detecting box 100 into and out of the nucleic acid detecting apparatus.
In this embodiment, the second casing 12 is provided with a plurality of through holes 17, and the through holes 17 can facilitate dissipation of heat in the case 1, thereby ensuring normal detection of the detection chip 2.
In this embodiment, a cover film 19 is disposed on the sample addition port 13 to seal the sample addition port 13.
Referring to fig. 2 to 4, and fig. 8 and 9, the electrophoresis cassette 3 includes an electrophoresis tank 31, electrophoresis electrodes 32 disposed at two ends of the electrophoresis tank 31, a gel medium 33 disposed between the two electrophoresis electrodes 32, and a liquid injection tank 34 disposed at one end of the gel medium 33. The electrophoresis electrode 32 is electrically connected to the external power source 7, the liquid injection groove 34 penetrates through the first housing 11 to be communicated with the liquid outlet 51, so as to realize the communication between the electrophoresis cassette 3 and the channel 5, and the nucleic acid amplification product combined with the fluorescent reagent enters the gel medium 33 through the liquid injection groove 34 in the liquid outlet region D, thereby performing the electrophoresis detection.
In this embodiment, the electrophoresis tank 31 includes a bottom plate 311 and a plurality of side walls 312 connected to the bottom plate 311, and the electrophoresis tank cover plate 35 is disposed at one end of the side walls 312 far from the bottom plate 311. The bottom plate 311 is a transparent plate, and the image capturing device can capture an image of the electrophoresis detection result at the outer side of the bottom plate 311.
In this embodiment, a sealing rubber ring (not shown) is disposed between the sidewall 312 and the bottom surface of the first casing 11 to improve the sealing performance of the electrophoresis cassette 3.
In another embodiment, an electrophoresis tank cover (not shown) is disposed on a side of the electrophoresis tank 31 close to the box body 1, wherein the electrophoresis tank cover is used for sealing the electrophoresis tank 31, and the liquid injection tank 34 penetrates the electrophoresis tank cover and is communicated with the liquid outlet 51.
In this embodiment, the electrophoresis tank 31 further includes a plurality of detents (not shown) disposed on the bottom plate 311, the gel medium 33 is substantially a rectangular parallelepiped and can be retained between the plurality of detents, and the detents are designed to prevent the gel medium 33 from moving and being misaligned, thereby ensuring the accuracy of electrophoresis detection.
Referring to fig. 3, in the present embodiment, the bottom plate 311 is a transparent glass plate, and the electrophoresis result can be observed.
In this embodiment, the number of the detents 313 is four, and the four detents 313 are located at four corners of the gel medium 33 having a rectangular parallelepiped structure, respectively, to fix the gel medium 33.
In the present embodiment, the gel medium 33 may be agar gel, agarose gel, or other gel for electrophoresis detection.
Referring to fig. 8 and fig. 9 in combination with fig. 7, in order to make the nucleic acid amplification product smoothly enter the electrophoresis cartridge 3, a first porous adsorption block 38 is disposed in the liquid injection groove 34, the first porous adsorption block 38 carries a wetting liquid 39, one end of the first porous adsorption block 38 extends into the bottom of the liquid injection groove 34, and the other end is flush with or protrudes from the surface of the liquid outlet 51 near the channel 5. When the nucleic acid amplification product moves to the liquid outlet region D, it contacts the first porous adsorption block 38, dissolves in the wetting liquid 39, and flows to the bottom of the liquid injection groove 34 through the pores of the first porous adsorption block 37 for electrophoresis detection. This application adopts the mode of dry-type electrophoresis, only need before the equipment with first porous adsorption piece 38 adsorb a small amount of wetting fluid 39 alright, need not to add a large amount of wetting fluids in electrophoresis tank 31, also need not to set up the capillary, need not to consider the equipment precision, simplified the equipment flow and the equipment degree of difficulty of nucleic acid detecting box 100, be favorable to moreover that the nucleic acid amplification product successfully gets into in annotating the cistern 34.
In this embodiment, the first porous adsorption block 38 may be a sponge or other porous material.
In another embodiment, referring to fig. 8 and fig. 10 in combination with fig. 7, a second porous adsorption block 36 is disposed in the liquid injection tank 34, the wetting liquid 39 is carried in the second porous adsorption block 36, an adsorption tube 52 is disposed at the liquid outlet 51, one end of the adsorption tube 52 is inserted into the second porous adsorption block 36, and the other end is flush with or protrudes from the surface of the liquid outlet 51 near the channel 5, and the adsorption tube 52 is filled with the wetting liquid 39 in the second adsorption block 36. When the nucleic acid amplification product moves to the liquid outlet region D, the nucleic acid amplification product contacts the adsorption tube 52, is dissolved in the wetting liquid 39, enters the second adsorption block 36 through the adsorption tube 52, and enters the bottom of the liquid injection groove 34 for electrophoresis detection. This application adopts the mode of dry-type electrophoresis, only need before the equipment with second porous adsorption block 36 adsorb a small amount of wetting liquid 39 alright, need not to add a large amount of wetting liquids in electrophoresis tank 31, need not to consider the equipment precision of adsorption tube 52, simplified the equipment flow and the equipment degree of difficulty of nucleic acid detection box 100, be favorable to moreover that the nucleic acid amplification product successfully gets into in annotating the cistern 34.
In another embodiment, a wetting solution (e.g., buffer) may be added to the injection tank 34, and the wetting solution is mainly used to enable the nucleic acid molecules in the nucleic acid amplification product to completely enter the gel medium 33.
Referring to fig. 4, each electrophoresis electrode 32 includes an electrode body 321 and electrode pads 322 electrically connected to the electrode body 321, and the two electrode pads 322 are disposed on the outer surface of the sidewall 312 of the electrophoresis tank 31, so as to be electrically connected to the external power source 7.
In the present embodiment, the material of the two electrode bodies 321 may be metal, or may be a composite structure of metal, anion resin, and cation resin.
In this embodiment, the electrode sheet 322 may be a metal sheet, specifically, a copper sheet.
In this embodiment, the electrophoresis cassette 3 further includes an electronic identification code 37 (specifically, a two-dimensional code) attached to the outer surface of the bottom plate 311 of the electrophoresis tank 31, and the image acquisition device can record the electrophoresis detection image by identifying the electronic identification code 37, so as to facilitate tracking of the subsequent detection result.
After the nucleic acid detecting cassette 100 is assembled, in actual use, the use process of the nucleic acid detecting cassette 100 includes the following steps:
in step S11, referring to FIG. 1, a sample addition part A of the detection chip 2 is injected with a detection solution a containing a nucleic acid sample through a sample addition port 13.
In step S12, referring to fig. 7, the detection solution a is driven to move to the nucleic acid amplification region C in the channel 5 according to a predetermined path by adjusting the voltage between the corresponding driving electrode 241 and the conductive layer 25 in the driving circuit 24, thereby completing the nucleic acid amplification reaction. Specifically, the number of nucleic acid amplification regions C is two, and the temperatures to be heated are 90 ℃ to 105 ℃ and 40 ℃ to 75 ℃, respectively.
In one embodiment, the specific nucleic acid amplification reaction process comprises, in order: firstly, performing thermal denaturation for 15-25min at the temperature of 90-105 ℃; secondly, RT reverse transcription is carried out for 5-15min under the condition of 45-60 ℃; thirdly, heating for 1-5min at the temperature of 90-100 ℃; and fourthly, heating for 20 to 50 seconds at the temperature of between 90 and 100 ℃ and heating for 40 to 60 seconds at the temperature of between 55 and 65 ℃, wherein the fourth step finishes the amplification reaction by circulating for 35 to 50 times (preferably 45 times). A temperature sensor and a time relay may be employed to sense the heating temperature and heating time.
In another embodiment, the specific nucleic acid amplification reaction process comprises, in order: firstly, performing thermal denaturation for 3-8min at the temperature of 90-105 ℃; secondly, proliferating for 3-8min at the temperature of 45-60 ℃; thirdly, heating for 3-8min at the temperature of 90-100 ℃; fourthly, amplifying for 3 to 8 seconds at the temperature of between 90 and 100 ℃, amplifying for 10 to 20 seconds at the temperature of between 50 and 65 ℃ and amplifying for 10 to 20 seconds at the temperature of between 68 and 75 ℃, wherein the fourth step finishes the amplification reaction by circulating for 35 to 50 times. Preferably, the specific nucleic acid amplification reaction process comprises, in order: firstly, performing thermal denaturation for 3-5min at the temperature of 95-97 ℃; secondly, proliferating for 3-5min at the temperature of 55-60 ℃; thirdly, heating for 3-8min at the temperature of 95-97 ℃; fourth, amplification is carried out for 3-5 seconds at 95-97 ℃, for 15-20 seconds at 55-60 ℃ and for 15-20 seconds at 70-72 ℃, wherein the fourth step is cycled for 43-45 times (preferably 45 times) to finish the amplification reaction.
Step S13, referring to fig. 7 and fig. 4, after amplification is completed, the nucleic acid amplification product is driven by the driving electrode 241 to move to the reagent storage area B, and the pressing member 6 is pressed to puncture the reagent pouch 6, so that the fluorescent reagent enters the reagent storage area B and is mixed with the nucleic acid amplification product, and the mixed fluorescent reagent enters the electrophoresis cassette 3 from the liquid outlet 51 of the liquid outlet area D.
Step S15, controls the electrophoresis cassette 3 to perform electrophoresis detection.
In this embodiment, since the nucleic acid detecting cassette 100 is a disposable product and one detecting cassette 20 is used for each sample, the detecting cassette 20 does not require a washing process.
In the present embodiment, the cartridge 20 has a substantially cubic structure.
Compared with the prior art, the nucleic acid detection box 100 integrates the electrophoresis box 3 and the detection chip 2, the detection liquid a can directly enter the electrophoresis box 3 for electrophoresis detection after completing nucleic acid amplification reaction in the detection chip 2, the process is smooth, equipment does not need to be replaced, and a professional does not need to perform sample transfer operation, so that the detection efficiency is greatly improved. Moreover, the nucleic acid detecting cassette 100 does not need to be provided with a heating device, so that the preparation difficulty and the preparation cost of the detecting chip 2 are greatly simplified. In addition, dry electrophoresis is adopted, on the premise of ensuring the accuracy of electrophoresis detection results, no buffer solution needs to be added, and the assembly process and the assembly difficulty are simplified.
Referring to fig. 11 and 12, the present invention further provides a nucleic acid detecting apparatus 200, wherein the nucleic acid detecting apparatus 200 includes a host 201 and the nucleic acid detecting cartridge 100 as described above, the host 201 is provided with at least one detecting cartridge mounting area 202, and the nucleic acid detecting cartridge 100 is detachably mounted in the detecting cartridge mounting area 202.
The host 201 further comprises a host heating device 204 and a host connector 203, wherein the host heating device 204 and the host connector 203 are both disposed in the test cassette mounting area 202. When the nucleic acid detecting cassette 100 is placed in the detecting cassette mounting region 202, the host connector 203 can extend into the connecting port 15 to abut against the control board 4 on the detecting chip 2 and realize electrical connection, and the host heating device 204 can extend into the heating port 14 to contact with the surface of the detecting chip 2, thereby heating the nucleic acid detecting cassette 100.
The host 201 further includes an image capturing device 205, the image capturing device 205 is disposed on a side of the detecting box mounting area 202 corresponding to the electrophoresis box 3, and the image capturing device 205 is configured to capture an image of the electrophoresis box 3.
In actual detection, mixing the collected nucleic acid sample of the subject with a detection reagent (e.g., buffer solution) to form a detection solution, and preheating the detection solution; after the detection liquid is preheated, the detection liquid is heated in the nucleic acid detection box 100, so that the detection liquid enters the nucleic acid detection box 100 to carry out nucleic acid amplification reaction and electrophoresis detection; the image acquiring device 205 acquires an image of the electrophoresis cassette 3 after the electrophoresis detection is completed. The image is a fluorescence photo of electrophoresis detection, and a nucleic acid detection result can be obtained according to the fluorescence photo.
Referring to FIG. 12, the cartridge mounting region 202 includes a mounting groove 206 with an inclined design, and specifically, one end of the mounting groove 206 near the sample port 13 of the nucleic acid detecting cartridge 100 is higher than one end of the mounting groove 206 far away from the sample port 13. Because a large amount of bubbles can be generated in the silicon oil in the detection chip 2 in the PCR reaction process, especially after heating, the generated bubbles can be aggravated, and if the generated bubbles are retained in the channel 5, the action path of the detection liquid a in the channel 5 can be blocked by the bubbles, so that the detection liquid a cannot move, and further the detection fails. Therefore, the mounting groove 206 is designed to be inclined, so that the nucleic acid detecting cassette 100 can be arranged obliquely, the sample application end of the nucleic acid detecting cassette 100 is higher than the end where the nucleic acid amplification reaction occurs, and the bubbles generated in the nucleic acid detecting cassette 100 can naturally move to a high position and be naturally discharged from the sample application end of the nucleic acid detecting cassette 100, thereby not obstructing the movement path of the detecting liquid a.
Referring again to FIG. 11, the nucleic acid detecting apparatus 200 further includes a display 207, wherein the display 207 is used for displaying the result of the nucleic acid detection and the set corresponding reaction parameters. The nucleic acid detecting apparatus 200 further comprises a camera 208, and the camera 208 is used for collecting information of the nucleic acid sample to be detected and recording the whole nucleic acid detecting process.
The image acquisition device 205 acquires an electrophoresis image of the electrophoresis cassette 3, processes the image through the image processor, displays the processed image on the display screen 207, and also uploads a detection result to a client for related personnel to review.
FIG. 13 is a schematic diagram of a test result obtained by using the nucleic acid detecting apparatus 200 according to the embodiment of the present invention. In this embodiment, by defining the range of each line on the standard fluorescent photograph in advance, the device can automatically recognize the detection result after the test result is obtained. Wherein, if the position of the marker of the first line is within the predefined range, it can be determined that the nucleic acid sample includes the human gene, and if the position of the marker of the first line is not within the predefined range, it can be determined that the nucleic acid sample includes the human gene. If the position of the marker on the second line is within the predefined range, it can be determined that the nucleic acid sample contains the RNA replicase, and if the position of the marker on the second line is not within the predefined range, it can be determined that the nucleic acid sample contains the RNA replicase. If the position of the marker on the third line is within the predetermined range, it can be determined that the nucleic acid sample contains the N protein, and if the position of the marker on the third line is not within the predetermined range, it can be determined that the nucleic acid sample contains the N protein.
Compared with the prior art, the nucleic acid detection equipment provided by the invention can integrate the nucleic acid amplification reaction and the electrophoresis detection of nucleic acid into one equipment through the matching of the host and the nucleic acid detection box, has a simple integral structure, is simple and convenient to detect and operate, has low requirement on the specialty in the operation process and high detection efficiency, and greatly reduces the detection cost; meanwhile, the flexibility of the detection process is strong, the detection process does not need to be carried out in a fixed laboratory, the detection equipment is portable, and community detection or family detection can be realized.
Claims (13)
1. A nucleic acid detecting cassette characterized by comprising:
a box body;
the detection chip is arranged in the box body and comprises a first cover plate, a spacing layer and a second cover plate, two opposite surfaces of the spacing layer are respectively adjacent to the first cover plate and the second cover plate, the first cover plate, the spacing layer and the second cover plate are arranged in an enclosing manner to form a channel, and the channel is used for bearing detection liquid so that the detection liquid can carry out nucleic acid amplification reaction in the channel to obtain a nucleic acid amplification product; and
the electrophoresis box is arranged outside the box body and communicated with the channel, and is used for carrying out electrophoresis detection on the nucleic acid amplification product.
2. The nucleic acid detecting cassette according to claim 1, wherein the detecting chip further comprises a driving circuit provided on a side of the first cover plate adjacent to the second cover plate, a conductive layer provided on a side of the second cover plate adjacent to the first cover plate, a first dielectric layer provided on a side of the driving circuit adjacent to the second cover plate, and a second dielectric layer provided on a side of the conductive layer adjacent to the first cover plate, wherein the driving circuit and the conductive layer are electrically connected to an external electrophoresis, and the channel is formed between the first dielectric layer and the second dielectric layer.
3. The nucleic acid detecting cassette according to claim 2, wherein the driving circuit includes at least one nucleic acid amplification region, the cassette body has at least one heating port corresponding to the nucleic acid amplification region, and the detecting chip is exposed from the heating port.
4. The nucleic acid detecting cassette according to claim 3, wherein the cassette body comprises an upper casing and a lower casing, and the heating port is provided in each of the upper casing and the lower casing in correspondence with the nucleic acid amplification region.
5. The nucleic acid detecting cassette according to claim 2, wherein the driving circuit includes a plurality of driving electrodes arranged in an array, the driving electrodes are connected to an external power source, and the driving electrodes are controlled to be electrically connected to or disconnected from the conductive layer, so that the detection solution moves between two adjacent driving electrodes.
6. The nucleic acid detecting cassette according to claim 5, further comprising a control board electrically connected to each of the driving electrodes and the conductive layer, wherein the control board is provided on a side surface of the first cover plate adjacent to the second cover plate, and the control board is located outside the channel, and the cassette body is provided with a connection port corresponding to the control board, and the control board is exposed from the connection port.
7. The nucleic acid detecting cassette according to claim 2, wherein the drive circuit further comprises a sample addition region, a reagent storage region, and a liquid discharge region, the liquid discharge region communicating with the electrophoresis cassette.
8. The nucleic acid detecting cassette according to claim 7, wherein a reagent bag is provided in the reagent storage region, a fluorescent reagent is provided in the reagent bag, the reagent bag extends out of the second cover plate, and the cassette body is provided with a reagent tank corresponding to the reagent storage region, the reagent bag being accommodated in the reagent tank.
9. The nucleic acid detecting cassette according to claim 1, wherein the electrophoresis cassette includes an electrophoresis tank, an electrophoresis tank cover plate covering the electrophoresis tank, two electrophoresis electrodes respectively disposed at two ends of the electrophoresis tank, a gel medium disposed between the two electrophoresis electrodes, and a liquid injection tank disposed at one end of the gel medium, each electrophoresis electrode is electrically connected to an external power source, the first cover plate has a liquid outlet corresponding to the liquid injection tank, and the liquid outlet is communicated with the liquid injection tank.
10. The nucleic acid detecting cassette according to claim 9, wherein a first porous adsorption block is disposed in the liquid injection tank, the first porous adsorption block carrying a wetting liquid therein, one end of the first porous adsorption block extending into the bottom of the liquid injection tank, and the other end of the first porous adsorption block being flush with or protruding from the surface of the liquid outlet near the channel.
11. The nucleic acid detecting cassette according to claim 9, wherein a second porous adsorption block is disposed in the liquid injection tank, a wetting liquid is carried in the second porous adsorption block, an adsorption tube is disposed at the liquid outlet, one end of the adsorption tube is inserted into the second porous adsorption block, the other end of the adsorption tube is flush with or protrudes from the surface of the liquid outlet close to the channel, and the adsorption tube is filled with the wetting liquid in the second porous adsorption block.
12. A nucleic acid detecting apparatus characterized by comprising:
a host;
a cartridge mounting section provided on the host computer, the cartridge mounting section being configured to detachably mount a nucleic acid detecting cartridge according to any one of claims 1 to 11.
13. The nucleic acid detecting apparatus according to claim 12, wherein the host includes:
the host connector is arranged in the detection box mounting area and is connected with the machine for electrically connecting the detection chip and the electrophoresis box;
the heating device is arranged in the detection box mounting area and is used for heating the detection chip; and
the image acquisition device is arranged in the detection box mounting area and corresponds to the electrophoresis box, and the image acquisition device is used for acquiring images of the electrophoresis box.
Priority Applications (4)
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| EP21199839.8A EP3978128A1 (en) | 2020-09-30 | 2021-09-29 | Nucleic acid detection kit and nucleic acid detection device |
| US17/488,660 US20220099621A1 (en) | 2020-09-30 | 2021-09-29 | Nucleic acid detection kit and nucleic acid detection device |
| JP2021160005A JP2022058275A (en) | 2020-09-30 | 2021-09-29 | Nucleic acid detection cassette and nucleic acid detection device |
| PCT/CN2021/122431 WO2022068937A1 (en) | 2020-09-30 | 2021-09-30 | Nucleic acid testing device |
Applications Claiming Priority (4)
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| US202063085368P | 2020-09-30 | 2020-09-30 | |
| US202063085385P | 2020-09-30 | 2020-09-30 | |
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| US63/085368 | 2020-09-30 |
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| CN202110604893.0A Pending CN114308147A (en) | 2020-09-30 | 2021-05-31 | Detection chip, nucleic acid detection box and nucleic acid detection equipment |
| CN202110602285.6A Pending CN114317220A (en) | 2020-09-30 | 2021-05-31 | Nucleic acid detecting cassette and nucleic acid detecting apparatus |
| CN202110604898.3A Pending CN114317250A (en) | 2020-09-30 | 2021-05-31 | Heating structure, detection chip, nucleic acid detection box and nucleic acid detection equipment |
| CN202110735245.9A Pending CN114317238A (en) | 2020-09-30 | 2021-06-30 | Nucleic acid detecting cassette and nucleic acid detecting apparatus |
| CN202110735243.XA Pending CN114317224A (en) | 2020-09-30 | 2021-06-30 | Nucleic acid detection host and nucleic acid detection equipment |
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| CN202110604893.0A Pending CN114308147A (en) | 2020-09-30 | 2021-05-31 | Detection chip, nucleic acid detection box and nucleic acid detection equipment |
| CN202110602285.6A Pending CN114317220A (en) | 2020-09-30 | 2021-05-31 | Nucleic acid detecting cassette and nucleic acid detecting apparatus |
| CN202110604898.3A Pending CN114317250A (en) | 2020-09-30 | 2021-05-31 | Heating structure, detection chip, nucleic acid detection box and nucleic acid detection equipment |
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| CN (5) | CN114308147A (en) |
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| CN114308147A (en) | 2022-04-12 |
| JP2022058179A (en) | 2022-04-11 |
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| TWI799879B (en) | 2023-04-21 |
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| TW202214835A (en) | 2022-04-16 |
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| TW202215032A (en) | 2022-04-16 |
| CN114317250A (en) | 2022-04-12 |
| TWI800863B (en) | 2023-05-01 |
| CN114317224A (en) | 2022-04-12 |
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