Nucleic acid detection device and nucleic acid detection method

Abstract

A nucleic acid detection device having a nucleic acid amplification function and a nucleic acid detection function that can be used at home is provided. [Solution] The present invention provides a nucleic acid detection device and a nucleic acid detection method. The nucleic acid detection device includes a carrier, a detector, and a mobile body. The carrier includes a substrate and a bridge, the bridge is installed on the upper surface of the substrate, and a sample pad is installed on the bridge. A channel is formed between the bridge and the substrate, and the mobile is removably installed in the channel, and the mobile includes at least one water-absorbing region and at least one non-water-absorbing region. The nucleic acid detection method is a method of performing nucleic acid amplification using a sample pad and performing nucleic acid detection using a nucleic acid detection device. The present invention solves the problems currently lacking in a nucleic acid detection device that can be used at home and has a nucleic acid amplification function and a nucleic acid detection function.

Description

[Technical field]

[0001] The present invention relates to the field of nucleic acid detection, and in particular to a nucleic acid detection device and a nucleic acid detection method. [Background technology]

[0002] Nucleic acid amplification and nucleic acid detection are widely used in in vitro diagnosis, life science research and other fields. With the development of science and technology, the devices used for nucleic acid amplification and nucleic acid detection have gradually begun to develop in the direction of miniaturization, low cost and convenience. However, the currently used nucleic acid amplification devices generally need to strictly control the temperature during nucleic acid amplification reaction, so they need to be equipped with a temperature control device, making it difficult to miniaturize and reduce the cost of the nucleic acid amplification device, and it is necessary to further perform nucleic acid detection after the completion of nucleic acid amplification to confirm the results of nucleic acid amplification. For the above reasons, many devices on the market are devices that only have a diffusion detection function, such as rapid virus screening, but in this type of rapid screening, the virus amount of the user is not high enough in the early stage, so it is easy to cause false negative problems. Therefore, currently, the market is lacking a nucleic acid detection device that can be used at home and has a nucleic acid amplification function and a nucleic acid detection function, which is a problem to be solved. Summary of the Invention [Problem to be solved by the invention]

[0003] One of the objects of the present invention is to solve the current problem of a shortage of nucleic acid detection devices having a nucleic acid amplification function and a nucleic acid detection function that can be used at home. [Means for solving the problem]

[0004] Based on the object of the present invention, the present invention provides a nucleic acid detection device comprising a carrier, a detector and a mobile body, the carrier comprising a substrate and a bridge section, the bridge section being installed on the upper surface of the substrate section, a channel being formed between the bridge section and the substrate section, the detector comprising an initiation pad, at least one control line and at least one detection line, the detector being installed on the upper surface of the substrate section, the initiation pad of the detector being located within the channel, the bridge section being provided with a window for viewing the detection result displayed by the at least one control line and at least one detection line of the detector, the bridge section being provided with a sample pad at a position corresponding to the initiation pad of the detector, and the mobile body being removably installed within the channel, comprising at least one water-absorbing region and at least one non-water-absorbing region.

[0005] In one embodiment of the present invention, the sample pad includes a nucleic acid adsorption layer.

[0006] In one embodiment of the present invention, the sample pad further includes a nucleic acid extraction layer laminated on the upper surface of the nucleic acid adsorption layer.

[0007] In one embodiment of the invention, the sample pad further comprises nucleic acid amplification reagents.

[0008] In one embodiment of the present invention, the nucleic acid detection device further includes a guide body, one end of which is connected to the upper surface of the movable body, the guide body covering the upper surface of the sample pad of the bridge portion of the carrier, a plurality of openings being provided on the guide body, each opening of the plurality of openings being exposed to the upper surface of the sample, at least one of the plurality of openings corresponding to the water-absorbing region of the movable body, and at least one of the plurality of openings corresponding to the non-water-absorbing region of the movable body.

[0009] In one embodiment of the present invention, at least one of the plurality of openings corresponding to the water-absorbing region of the mobile body is further provided with a nucleic acid extraction layer.

[0010] The present invention provides a nucleic acid detection method including a nucleic acid detection device providing step, a sample adding step, a nucleic acid amplifying step and a nucleic acid detecting step in that order. The nucleic acid detection device providing step provides a nucleic acid detection device. The sample adding step corresponds the water absorbing region of the mobile body to the sample pad, and then adds a test sample containing a target nucleic acid to the sample pad. The nucleic acid amplifying step corresponds the non-water absorbing region of the mobile body to the sample pad, and then causes the target nucleic acid in the sample pad to react with a nucleic acid amplification reagent to form an amplicon of the target nucleic acid. The nucleic acid detecting step removes the mobile body from the channel, presses the bridge part to bring the sample pad into contact with the initiation pad, adds an eluent to the sample pad, and causes the amplicon of the target nucleic acid in the sample pad to flow together with the eluent to the initiation pad of the detection body, and then flows from the initiation pad to the at least one control line and the at least one detection line of the detection body, and the amplicon of the target nucleic acid is captured by the at least one detection line.

[0011] In one embodiment of the present invention, after the sample addition step is completed, a first cleaning step is further included, in which the first cleaning step corresponds to the water absorption area of ​​the mobile body with the sample pad, and then a first cleaning liquid is added to the sample pad.

[0012] In one embodiment of the present invention, after the nucleic acid amplification process is completed, a second washing process is further included, in which the water absorption region of the mobile body is aligned with the sample pad, and then a second washing solution is added to the sample pad.

[0013] In one embodiment of the present invention, the method further includes a first washing step after completion of the sample addition step, and a second washing step after completion of the nucleic acid amplification step, in which the first washing step is to add a first washing liquid to the sample pad after matching the absorbent region of the mobile body to the sample pad, and the second washing step is to add a second washing liquid to the sample pad after matching the absorbent region of the mobile body to the sample pad. Effect of the Invention

[0014] In summary, the nucleic acid detection device and nucleic acid detection method provided by the present invention enable users to easily complete the entire process from adding the test sample and amplifying and detecting nucleic acid in a home environment, thereby solving the problem currently lacking in nucleic acid detection devices that can be used at home and have nucleic acid amplification and detection functions. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a perspective view of an embodiment of a nucleic acid detection device. [Diagram 2] FIG. 2 is a top view of FIG. [Diagram 3] FIG. 3 is an explanatory diagram showing a state in which a guide body is removed from FIG. 2. [Figure 4] FIG. 4 is an explanatory diagram showing a state in which the bridge portion and the moving body are removed from FIG. 3. [Diagram 5] 2 is a perspective view of a support and a detection body in FIG. 1. [Figure 6] 3 is a cross-sectional view taken along the line AA in FIG. 2. [Figure 7] FIG. 2 is a side view of a first embodiment of a carrier. [Figure 8] FIG. 4 is a side view of a second embodiment of the carrier. [Figure 9] FIG. 11 is a side view of a third embodiment of the carrier. [Figure 10] FIG. 11 is a side view of a fourth embodiment of the carrier. [Figure 11] FIG. 1 is a process diagram of a nucleic acid detection method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The term "one embodiment" as used throughout the specification means that a particular advantage, structure, or feature described in connection with the embodiment is included in at least one embodiment. Thus, references to "one embodiment" in various places in the specification do not necessarily all refer to the same embodiment. Also, the particular advantages, structures, or features may be combined in any manner in one or more embodiments.

[0017] References such as "first," "second," "third," "fourth," "fifth," and "sixth" appearing throughout the specification are for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0018] 1, 4, 5, 6, 7, 8, 9 and 10, the present invention provides a nucleic acid detection device 1 including a carrier 2, a detector 3 and a mobile body 4. The carrier 2 includes a substrate portion 20 and a bridge portion 22. The bridge portion 22 is installed on the upper surface of the substrate portion 20, and a channel 222 is formed between the bridge portion 22 and the substrate portion 20. The detector 3 includes an initiation pad 30, at least one control line 340 and at least one detection line 342. The detector 3 is installed on the upper surface of the substrate portion 20 of the carrier 2, and the initiation pad 30 of the detector 3 is located in the channel 222. A window 224 is provided at one end of the bridge portion 22, and the window 224 is used to pass the detector 3 to one end of the bridge portion 22. A sample pad 220 is provided at a position of the bridge portion 22 corresponding to the initiation pad 30 of the detector 3. The mover 4 is removably mounted within the channel 222 and includes at least one water-absorbing region and at least one non-water-absorbing region B.

[0019] 1, 2, 3, 5, 6 and 7, in one embodiment of the present invention, the substrate part 20 of the carrier 2 is used to carry the bridge part 22, the detection body 3 and the moving body 4. The bridge part 22 includes a first side wall 22a, a connection surface 22b and a second side wall 22c, one side of the first side wall 22a is connected to the upper surface of the substrate part 20 and is adjacent to the position of the starting pad 30, the other side of the first side wall 22a is connected to one side of the connection surface 22b, the other side of the connection surface 22b is connected to one side of the second side wall 22c, the other side of the second side wall 22c is connected to the upper surface of the substrate part 20 and is adjacent to the position of the at least one control line 340 and the at least one control line 340, and a window 224 is provided at the connection point between the second side wall 22c and the substrate part 20, and the window 224 allows the detection body 3 to pass through the second side wall 22c. A sample pad 220 is provided on the connection surface 22b at a location corresponding to the start pad 30 of the detection element 3.

[0020] 1, 2, 3 and 4, in one embodiment of the present invention, the detection body 3 is a lateral flow immunochromatography test paper, and the detection body 3 is fixed to the upper surface of the substrate part 2 of the support 2 by adhesion. The detection body 3 includes an initiation pad 30, a binding pad 32, a reaction membrane 34 and an absorption pad 36, and the detection body 3 is provided with the initiation pad 30 at one end and the absorption pad 36 at the other end, one end of the initiation pad 30 is connected to one end of the binding pad 32, the other end of the binding pad 32 is connected to one end of the reaction membrane 34, and the other end of the reaction membrane 34 is connected to one end of the absorption pad 36. The reaction membrane 34 includes at least one control line 340 and at least one detection line 342.

[0021] 1, 2, 3, 4, 5, 6 and 7, in one embodiment of the present invention, the detection body 3 passes through the second side wall 22c through the window 224, so that the at least one control line 340 and the at least one detection line 342 of the detection body are located outside the bridge portion 22, allowing the user to see the at least one control line 340 and the at least one detection line 342 of the detection body 3. Therefore, the main function of the window 224 provided in the bridge portion 22 is to allow the user to see the at least one control line 340 and the at least one detection line 342 of the detection body 3, thereby making it easier for the user to read the detection result.

[0022] The above-mentioned embodiment of the support 2 does not limit the support 2 to the above embodiment, and hereinafter, the above-mentioned embodiment of the support 2 will be referred to as the first embodiment of the support 2, and other embodiments of the support 2 will be separately exemplified, respectively representing examples of the second, third, and fourth embodiments of the support 2. The differences between the second, third, and fourth embodiments of the support 2 and the first embodiment of the support 2 lie in the structure of the bridge portion 22 and the position where the window 224 is located on the bridge portion, and each embodiment of the support 2 will be described below with reference to Figs. 1 to 10.

[0023] Please refer to FIG. 7, which is a side view of a first embodiment of the support 2, for details please refer to the previous paragraph and further to FIGS. 1 to 6.

[0024] Referring to Figure 8, Figure 8 is a side view of the second embodiment of the support 2, in which one end and the other end of the bridge portion 22 are both installed on the upper surface of the substrate portion 20, and the difference between the second embodiment and the first embodiment of the support 2 is that the bridge portion 22 covers the entire detection body 3, a sample pad 220 is provided at a position on the bridge portion 22 corresponding to the start pad 30 of the detection body 3, and a window 224 is provided at a position on the bridge portion 22 corresponding to at least one control line 340 and at least one detection line 342 of the detection body 3.

[0025] Referring to Figure 9, Figure 9 is a side view of the third embodiment of the support 2, which is basically the same as the second embodiment of the support 2, with the difference being that the location where the sample pad 220 of the bridge portion 22 is provided is in the form of a groove, thereby shortening the distance between the sample pad 220 and the starting pad 30 of the detection body 3.

[0026] 10, which is a side view of the fourth embodiment of the carrier 2. One end of the bridge portion 22 is installed on the upper surface of the substrate portion 20, the bridge portion 22 is stepped, and shortens the distance between the sample pad 220 and the start pad 30 of the detection body 3, the other end of the bridge portion 22 is provided with a first fastener Bu1, and one end of the substrate portion 20 is provided with a second fastener Bu2, the first fastener Bu1 is used to engage with the second fastener Bu2, and when the first fastener Bu1 and the second fastener Bu2 engage with each other, the bridge portion 22 approaches the sample pad 220 and brings the sample pad 220 into contact with the start pad 30 of the detection body 3.

[0027] In the above-mentioned embodiment of each carrier 2, the window 224 described in the first embodiment of the carrier 2 is an opening. In the second, third and fourth embodiments of the carrier 2, the embodiment of the window 224 may be an opening or a light-transmitting material, and examples of the light-transmitting material include transparent plastic, plastic wrap or a transparent glass plate, but in actual implementation, it is not limited to this and may be other light-transmitting materials, as long as the user can view the detection result displayed by at least one control line 340 and at least one detection line 342 of the detection body 3 from the bridge part 22.

[0028] 1, 2, 3, 5 and 6, in one embodiment of the present invention, the moving body 4 includes a moving substrate 40, the moving substrate 40 includes at least one water-absorbent region and at least one non-water-absorbent region B, the water-absorbent region refers to the region where an absorbent pad is provided on the moving body 4, the non-water-absorbent region B refers to the region where a waterproof pad WP is provided on the moving body 4, the absorbent pad and the waterproof pad WP are both installed on the upper surface of the moving substrate 40, the moving body 4 moves along the direction of the channel 222, and the water-absorbent region of the moving body 4 corresponds to the sample pad 220 or the non-water-absorbent region B of the moving body 4 corresponds to the sample pad 220, and the sample pad 220 can be stacked on the upper surface of the absorbent pad or the waterproof pad WP by the movement of the moving body 4, and the moving body 4 can also be taken out of the channel 222. The above is merely an example, and in actual implementation, the present invention is not limited thereto, and the non-absorbent area B of the moving body 4 may refer to an area where the sample pad 220 is not placed, so that when the non-absorbent area B corresponds to the sample pad 220, the sample pad 220 does not contact the absorbent pad, and therefore the liquid added to the sample pad 220 remains within the sample pad 220. The method of moving the moving body 4 may be to pinch the part of the moving body 4 outside the channel 222 with hands or use a clamp tool to push or pull the moving body 4, and move the moving body 4 along the direction of the channel 222, and the clamp tool may be, for example, tweezers or a binder clip, and the present invention is not particularly limited thereto.

[0029] 6, in one embodiment of the present invention, the sample pad 220 includes a nucleic acid extraction layer 2200 and a nucleic acid adsorption layer 2202, and the nucleic acid extraction layer 2200 is laminated on the upper surface of the nucleic acid adsorption layer 2202. In another embodiment of the present invention, the sample pad 220 includes only the nucleic acid adsorption layer 2202.

[0030] Referring to Figures 1, 2, 5 and 6, in one embodiment of the present invention, the nucleic acid detection device 1 further includes a guide body 5, one end of the guide body 5 is connected to the upper surface of the moving body 4, and when the moving body 4 is placed in the channel 222, the guide body 5 covers the upper surface of the sample pad 220, and the guide body 5 is provided with a plurality of openings that expose the upper surface of the sample pad 220, at least one of the plurality of openings corresponds to the water-absorbing region of the moving body 4, and at least one of the plurality of openings corresponds to the non-water-absorbing region B of the moving body 4.

[0031] Referring to Figures 1, 2 and 6, in one embodiment of the present invention, at least one of the multiple openings of the guide body 5 corresponding to the water absorption region of the mobile body 4 is further provided with a nucleic acid extraction layer 2200, and the other openings of the multiple openings are used to expose the upper surface of the sample pad 220.

[0032] Referring to Figures 1, 2, 3, 5 and 6, in one embodiment of the present invention, the method further includes a plurality of prompt marks, which are marked on the guide body 5 and the support 2, and the prompt marks are marks indicating the process sequence of the nucleic acid detection method of the present invention, and may be character marks, pattern marks or combinations thereof, and the marking method of each prompt mark may be printing, embossing, punching, seal marking or highlighter marking, and the present invention is not particularly limited. When multiple prompt marks are marked by punching, the connection surface 22b of the bridge portion 22 may further include a guide mark 6, which, in combination with the multiple prompt marks, indicates to the user the position of the moving body 4 to which the current sample pad 220 corresponds, and the guide mark 6 may be a character mark, a pattern mark, or a combination thereof, and may be a printing, embossing, punching, seal marking or highlighter marking method, so that when the prompt mark is not overlapped with the guide mark 6, the guide mark 6 is covered under the guide body 5, and when the prompt mark overlaps with the guide mark 6, the prompt mark exposes the guide mark 6.

[0033] 1 to 11, the present invention provides a nucleic acid detection method including a nucleic acid detection device providing step S10, a sample adding step S20, a nucleic acid amplifying step S30, and a nucleic acid detecting step S40 in this order. The nucleic acid detection device providing step S10 provides the nucleic acid detection device 1 of the present invention. The sample adding step S20 corresponds the water absorbing region of the mobile body 4 to the sample pad 220, and then adds a test sample to the sample pad 220, and the test sample contains a target nucleic acid. The nucleic acid amplifying step S30 corresponds the non-water absorbing region B of the mobile body 4 to the sample pad 220, and then causes the target nucleic acid in the sample pad 220 to react with a nucleic acid amplification reagent to form an amplicon of the target nucleic acid. The nucleic acid detection process S40 involves removing the mobile body 4 from the channel 222, pressing the bridge portion 22 to bring the sample pad 220 into contact with the initiation pad 30, adding an eluent to the sample pad 220, and causing the amplicon of the target nucleic acid in the sample pad 220 to flow together with the eluent to the initiation pad 30 of the detection body 3, and then flowing from the initiation pad 30 to the at least one control line 340 of the detection body 3 and the at least one control line 340 described above, and the amplicon of the target nucleic acid is captured at at least one detection line 342.

[0034] 1 to 11, in one embodiment of the present invention, after the sample addition step S20 is completed, a first cleaning step S25 is further included, in which the water absorption area of ​​the mobile body 4 is aligned with the sample pad 220 and then a first cleaning liquid is added to the sample pad 220.

[0035] 1 to 11, in one embodiment of the present invention, after the nucleic acid amplification step S30 is completed, a second washing step S35 is further included, in which the water absorption area of ​​the mobile body 4 is aligned with the sample pad 220 and then a second washing liquid is added to the sample pad 220.

[0036] 1 to 11, one embodiment of the present invention further includes a first washing step S25 after completion of the sample adding step S20, and further includes a second washing step S35 after completion of the nucleic acid amplifying step S30. The first washing step S25 is a step of adding a first washing liquid to the sample pad 220 after the water absorbing region of the mobile body 4 corresponds to the sample pad 220, and the second washing step S35 is a step of adding a second washing liquid to the sample pad 220 after the water absorbing region of the mobile body 4 corresponds to the sample pad 220.

[0037] 1 to 11, in one embodiment of the present invention, the "reacting the target nucleic acid in the sample pad 220 with the nucleic acid amplification reagent" described in the nucleic acid amplification step S30 means adding the nucleic acid amplification reagent to the sample pad 220, causing the nucleic acid amplification reagent to react with the target nucleic acid in the sample pad 220, and forming an amplicon of the target nucleic acid. In another embodiment of the present invention, the "reacting the target nucleic acid in the sample pad 220 with the nucleic acid amplification reagent" described in the nucleic acid amplification step S30 means that the sample pad 220 further contains the nucleic acid amplification reagent, and the nucleic acid amplification reagent can be held in the sample pad 220 in a freeze-dried state, so that after the sample addition step S20 is completed, the non-water-absorbent region B of the mobile body 4 is made to correspond to the sample pad 220, and the target nucleic acid in the sample pad 220 is caused to react with the nucleic acid amplification reagent in the sample pad 220 to form an amplicon of the target nucleic acid.

[0038] In the following, each embodiment will be described in detail with reference to the accompanying drawings. The specific contents of each embodiment will be described in the following paragraphs. The embodiments include embodiment 1, embodiment 2 and embodiment 3.

[0039] In the following examples, Agrobacterium (scientific name: Agrobacterium tumefacien) bacterial liquid or Agrobacterium genomic nucleic acid is given as an example of a test sample, but in actual practice, the test sample is not limited to this, and the test sample may be an organism such as a microbial individual, tissue, cell, bacteria, fungus, algae, protozoa, or protozoan fungus, or a virus. The test sample may also be free nucleic acid, and the free nucleic acid may be DNA or RNA, and the present invention is not particularly limited, and the free nucleic acid may be nucleic acid formed by nucleic acid extraction from an organism such as a microbial individual, tissue, cell, bacteria, fungus, algae, protozoa, or protozoan fungus, or a virus, and examples of nucleic acid extraction include liberating nucleic acid from the organism or virus using a lysis solution, or adding the organism or virus to a membrane layer containing a dry lysis solution to liberate nucleic acid, and examples of the membrane layer include glass fiber membrane, filter paper, gel, nonwoven fabric, cellulose membrane, polyester membrane, polypropylene membrane, and the like, and the present invention is not particularly limited.

[0040] In each of the following examples, the target nucleic acid is taken as an example of the virG gene of Agrobacterium, and the nucleotide sequence of the virG gene of Agrobacterium is as shown in SEQ ID NO: 1. Please refer to Table 1 below, which includes the sequence name and the corresponding nucleotide sequence, but is not limited thereto in actual practice. The target nucleic acid to be detected can be selected according to practical needs. The present invention is not limited thereto, and the target nucleic acid may be either DNA or RNA.

[0041] Referring to FIG. 4 and FIG. 6, in the following examples, the nucleic acid amplification reaction uses a recombinase polymerase amplification technique, so the nucleic acid amplification reagent is a recombinase polymerase amplification (RPA) reagent, which contains a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing polymerase (SdP). The primers include a nucleotide sequence of SEQ ID NO: 1, a forward primer, and a reverse primer, the forward primer and the reverse primer are used to bind to SEQ ID NO: 1 and serve as the starting point for nucleic acid amplification, the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 2, see Table 1 below, which shows the sequence name and the corresponding nucleotide sequence, the 5' end of the nucleotide sequence of the forward primer is connected to a chemical marker, the chemical marker is biotin, the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 3, see Table 1 below, which shows the sequence name and the corresponding nucleotide sequence, and the 5' end of the reverse primer is connected to a fluorescent marker, the fluorescent marker is fluorescein isothiocyanate,FITC), after the subsequent nucleic acid amplification reaction is carried out on the target nucleic acid, the forward primer and reverse primer remain on the amplicon of the target nucleic acid, and the amplicon of the target nucleic acid is provided with a fluorescent marker and a chemical marker, which facilitates the subsequent detection of the amplicon of the target nucleic acid by the detector 3. Since the amplicon of the target nucleic acid is provided with a fluorescent marker, it can be judged whether the detection line 342 of the detector 3 generates a fluorescent signal in the subsequent stage. If a fluorescent signal is generated, it indicates that the amplicon of the target nucleic acid has been detected, but in actual practice, the present invention is not limited to this, the fluorescent marker can be a luminescent material for other commonly used primer marking, and the chemical marker can be a chemical base modification for other commonly used primer marking, and the forward primer and reverse primer can be designed accordingly according to the target material to be detected, and in actual practice, the forward primer or reverse primer is bound to recognize the homologous DNA sequence on the target nucleic acid in the test sample, and at the same time, the forward primer can be marked with a fluorescent marker and the reverse primer can be marked with a chemical marker, and the present invention is not particularly limited. Here, the recombinase binds to the forward primer or reverse primer, identifies the homologous DNA sequence on the target nucleic acid in the test sample, and simultaneously induces the forward primer or reverse primer to form a D-loop and a single-stranded DNA section. The single-stranded DNA binding protein binds to the single-stranded DNA section to stabilize the structure. Then, the strand displacement polymerase uses the position where the forward primer or reverse primer is bound as a starting point to perform a nucleic acid amplification reaction. By repeating the above-mentioned nucleic acid amplification reaction, the amplicon of the target nucleic acid is continuously generated, but in actual implementation, it is not limited thereto. The nucleic acid amplification reaction may be performed by a technique such as Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HDA), etc.The nucleic acid amplification reagent may be an isothermal nucleic acid amplification technique such as HDA (hardware assay), strand displacement amplification (Strand Displacement Amplification) (SDA), Nicking Enzyme Amplification Reaction (NEAR), Nucleic acid sequence-Based amplification (NASBA) technology, exponential amplification reaction (EXPAR) technology, or loop mediated isothermal amplification (LAMP) technology. According to the isothermal nucleic acid amplification technique used, a corresponding isothermal nucleic acid amplification reagent is used, that is, a strand displacement amplification reagent, a nicking enzyme amplification reaction reagent, a nucleic acid sequence-dependent amplification reagent, an exponential amplification reaction reagent, or a loop mediated isothermal amplification reagent, and at the same time, if the test sample is RNA, or if the test sample is cleaved by the nucleic acid extraction layer 2200 to generate RNA, a nucleic acid reverse transcription reaction reagent can be further added to the nucleic acid amplification reaction.

[0042] Table 1: Nucleotide sequences corresponding to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The left to right direction of the nucleotide sequence is from the 5' end to the 3' end. [Table 1]

[0043] Referring to FIG. 6, in each of the following embodiments, the nucleic acid extraction layer 2200 is a glass fiber membrane containing a dry dissolving solution, and the dissolving solution is a bacterial dissolving solution, but is not limited thereto in actual implementation. The nucleic acid extraction layer 2200 may be a filter paper, a gel, a nonwoven fabric, a cellulose membrane, a polyester membrane, or a polypropylene membrane. The dissolving solution may be other types of dissolving solutions, which can be adjusted according to the test sample, and dissolve other types of test samples, such as cells, viruses, fungi, protozoa, and algae, and release nucleic acids into the test sample.

[0044] Referring to Figure 6, in each of the following examples, nucleic acid adsorption layer 2202 is a silica membrane, but is not limited to this in actual implementation, and nucleic acid adsorption layer 2202 may be a film having silica particles, a film having diatomaceous earth, filter paper, Whatman 903 filter paper, cellulose membrane, Fusion 5 filter paper, polyethersulfone (polyethersulfone / PES) membrane, nylon membrane, etc.

[0045] In each of the following examples, the first cleaning liquid and the second cleaning liquid are both absolute ethanol, but in actual implementation, this is not limited to this, and the first cleaning liquid and the second cleaning liquid may be isopropyl alcohol or 95% v / v ethanol, or the first cleaning liquid and the second cleaning liquid may be different cleaning liquids, for example, the first cleaning liquid is isopropyl alcohol and the second cleaning liquid is absolute ethanol.

[0046] 1, 4, 5, 6 and 7, in the following examples, the support 2 of the nucleic acid detection device 1 is the first embodiment described above. As an example of the support 2, the substrate 20 is a rectangular piece body, and the first side wall 22a, the connection surface 22b and the second side wall 22c of the bridge portion 22 jointly form a U-shaped piece body, so that a channel 222 is formed between the substrate 20 and the bridge portion 22, the start pad 30 of the detection body 3 is located in the channel 222, the sample pad 220 is provided on the connection surface 22b of the bridge portion 22, the directly below the sample pad 220 corresponds to the start pad 30 of the detection body 3, the other end of the detection body 3 passes through the second side wall 22c through a window 224, and the bottom edge of the window 224 is connected to the upper surface of the substrate 20. The detection body 3 can be placed flat on the upper surface of the substrate portion 20, and the control line 340 and the detection line 342 are positioned outside the channel 222, making it easy for the user to observe, but in actual implementation, this is not limited to this, and the shape of the substrate portion 20 may be other shapes such as circular or square, and can be adjusted according to actual needs, and the present invention is not particularly limited, and the bridge portion 22 may be an arc-shaped piece body formed by the first side wall 22a, the connection surface 22b and the second side wall 22c jointly connected, or may be other structures that can similarly form a bridge, and any equivalent modification or change that can form a channel 222 between the substrate portion 20 should be considered to belong to the scope of the present invention. The material of the bridge portion 22 is white cardboard among paper materials, but is not limited thereto in actual implementation, and may be other types of paper materials such as laminated paper, craft paper, drawing paper, etc., and the bridge portion 22 may be a flexible material such as flexible plastic, flexible glass, or rubber, and the flexible plastic may be, for example, polyethylene (PE) or polyethylene terephthalate (PET). Since the bridge portion 22 is bendable, after the moving body 4 is removed from the channel 222, the first side wall 22a and the second side wall 22c of the bridge portion 22 are bent by pressing the connection surface 22b, or the connection surface 22b of the bridge portion 22 is bent toward the start pad 30, so that the sample pad 220 of the connection surface 22b can be brought into contact with the start pad 30 of the detector 3.Here, the sample pad 220 is fixed on the connection surface 22b by an embedding method, and the embedding method described here involves providing a through-hole at one end of the mobile body 4, fixing the sample pad 220 in the through-hole by adhesion, and exposing the top and bottom surfaces of the sample pad 220 at the same time, but in actual implementation, this is not limited to the above, and the sample pad 220 may be fixed in the through-hole by using double-sided tape, foam tape, or by fixing with a clamp. The sample pad 220 includes a nucleic acid extraction layer 2200 and a nucleic acid adsorption layer 2202, and the nucleic acid extraction layer 2200 is laminated on the top surface of the nucleic acid adsorption layer 2202.

[0047] 4, 6 and 7, in each of the following examples, the detection body 3 is a lateral flow immunochromatography paper, and is fixed to the upper surface of the support 2 by adhesion. The detection body 3 includes an initiation pad 30, a binding pad 32, a reaction membrane 34 and an absorption pad 36, the initiation pad 30 is provided at one end of the detection body 3, the absorption pad 36 is provided at the other end of the detection body 3, one end of the initiation pad 30 is laminated on one end of the binding pad 32, the other end of the binding pad 32 is laminated on one end of the reaction membrane 34, and one end of the absorption pad 36 is laminated on the other end of the reaction membrane 34. The initiation pad 30 is used to receive the eluent and the amplicon of the target nucleic acid, and allows the amplicon of the target nucleic acid to flow to the binding pad 32 together with the eluent. In the following examples, the nucleic acid adsorption layer 2202 is a silica film, and the silica film adsorbs the amplicon of the target nucleic acid, so that the pH value or salt concentration of the silica film can be changed by the eluent in the subsequent steps. The amplicon of the target nucleic acid is released from the silica film and flows to the initiation pad 30 of the detector 3 along with the flow of the eluent. The eluent is nuclease-free water, but is not limited to this in actual implementation. For example, the eluent is a TE buffer (Tris-EDTA buffer, TE buffer) with a pH value of 8.0. The TE buffer contains trishydroxymethylaminomethane (Tris) and ethylenediaminetetraacetic acid (ethylenediaminetetraacetic acid, In the low-salt buffer, the concentration of tris(hydroxymethylaminomethane) may be 10 mM, and the concentration of ethylenediaminetetraacetic acid may be 1 mM, but in actual practice, the concentration and pH value of each component in the low-salt buffer can be adjusted according to practical needs, and the low-salt buffer is not limited to TE buffer, and may be other buffers used for eluting nucleic acids.

[0048] Referring to FIG. 4, in the following embodiments, the initiation pad 30 is a glass fiber membrane, but in actual implementation, it is not limited thereto, and may be a filter paper, a nonwoven fabric, a cellulose membrane, a polyester membrane, or a polypropylene membrane.

[0049] Referring to FIG. 4, in each of the following embodiments, the binding pad 32 includes a chromogenic substance, the binding pad 32 is a glass fiber membrane, the chromogenic substance is a carrier particle of an anti-chemical marker antibody, the binding pad 32 is a glass fiber membrane, the chromogenic substance includes an anti-chemical marker antibody and a carrier particle, the anti-chemical marker antibody is connected to the surface of the carrier particle, the carrier particle is an anti-biotin antibody, and the carrier particle is a gold nanoparticle. Since the amplicon of the target nucleic acid is provided with a chemical marker, the anti-chemical marker antibody in the chromogenic substance binds to the chemical marker on the target nucleic acid, but is not limited to this in actual implementation, and the binding pad 32 may be a filter paper, a nonwoven fabric, a cellulose film, a polyester film, or a polypropylene film. The carrier particles in the chromogenic material are not limited to gold nanoparticles, and may be carriers such as fluorescent microbeads, colored polystyrene microbeads, magnetic microbeads, carbon nanoparticles, selenium nanoparticles, or silver nanoparticles. The surface of the carrier particles in the chromogenic substance is not limited to being connected to the anti-chemical marker antibody, and may be an anti-chemical marker nucleic acid aptamer, an anti-fluorescent marker antibody, or an anti-fluorescent marker nucleic acid aptamer. In another embodiment of the present invention, the chromogenic material may be an organic fluorescent group or a textile dye.

[0050] Referring to FIG. 4, in each of the following examples, the reaction membrane 34 is a nitrocellulose membrane, and the eluent flowing through the binding pad 32 is moved toward the absorption pad 36 by utilizing capillary action, and the amplicon of the target nucleic acid and the coloring substance also move toward the absorption pad 36 together with the eluent. However, in actual implementation, this is not limited to this, and the reaction membrane 34 may be a poly(vinylidene fluoride) membrane (PVDF), cellulose acetate, or nylon membrane.

[0051] Referring to FIG. 4, in each of the following examples, the number of detection lines 342 of the reaction membrane 34 is one, the detection line 342 includes an anti-fluorescent marker antibody, the anti-fluorescent marker antibody is an anti-fluorescent isothiocyanate antibody, the amplicon of the target nucleic acid is provided with a fluorescent marker, the amplicon of the target nucleic acid is captured by the anti-fluorescent marker antibody of the detection line 342 and fixed on the detection line 342, and a color-producing substance is bound to the target nucleic acid, so that a visually observable colored band is formed, but this is not limited to this in actual implementation, the number of detection lines 342 of the reaction membrane 34 may be multiple, and what is included in the detection line 3442 is not limited to an anti-fluorescent marker antibody, but can be adjusted according to the marker on the target nucleic acid to be captured, and is not limited to an antibody, but may be a nucleic acid aptamer, for example, the anti-fluorescent marker antibody may be an anti-chemical marker antibody, an anti-fluorescent marker nucleic acid aptamer, or an anti-chemical marker nucleic acid aptamer, etc.

[0052] Referring to FIG. 4, in each of the following examples, the number of control lines 340 of the reaction membrane 34 is one, the control line 340 includes a secondary antibody, which is used to capture the anti-chemical marker antibody in the chromogenic substance, so that the chromogenic substance can be fixed on the control line 340 and form a visually observable colored band, but this is not limited to this in actual implementation, and the number of control lines 340 of the reaction membrane 34 may be, for example, multiple, and the secondary antibody on the control line 340 of the reaction membrane 34 can be adjusted according to the form of the chromogenic substance to be captured, and may be an anti-fluorescent marker antibody, an anti-chemical marker nucleic acid aptamer, or an anti-fluorescent marker nucleic acid aptamer that can capture the chromogenic substance.

[0053] Referring to FIG. 4, in each of the following examples, the absorbent pad 36 is made of a highly absorbent paper material, such as filter paper that absorbs the eluent flowing from the reaction membrane 34 to the absorbent pad 36. However, in actual practice, the absorbent pad 36 is not limited to this, and may be made of a highly absorbent material such as nonwoven fabric or absorbent gel.

[0054] 1, 3, 5 and 6, in each of the following embodiments, the movable base 40 of the movable body 4 is a rectangular piece body, and two absorbent pads and one waterproof pad WP are provided on the movable base 40, and the two absorbent pads are respectively called the first absorbent pad AP1 and the second absorbent pad AP2, and the waterproof pad WP is installed between the first absorbent pad AP1 and the second waterproof pad WP, ​​and the area on the movable body 4 where the first absorbent pad AP1 is installed and the area where the second absorbent pad AP2 is installed are both water absorbing areas, and the movable body The area on the movable body 4 where the waterproof pad WP is installed is the non-absorbent area B, the area on the movable body 4 where the first absorbent pad AP1 is installed is called the first absorbent area A1, and the area on the movable body 4 where the second absorbent area AP2 is installed is called the second absorbent area A2, and the absorbent pad and waterproof pad WP are both installed on the upper surface of the movable body 4 by adhesion, the movable body 4 is removably installed in the channel 222, and since the movable body 4 is installed on the upper surface of the substrate portion 20, the part of the movable body 4 located within the channel 222 is laminated on the upper surface of the detection body 3. When the moving body 4 is removably installed in the channel 222, the moving body 4 can move along the direction of the channel 222, and even if the first water absorption area A1, the non-water absorption area B, or the second water absorption area A2 of the moving body 4 corresponds to the sample pad 220 and the sample pad 220 is stacked on the upper surface of the first absorbent pad AP1, the upper surface of the waterproof pad WP, ​​or the upper surface of the second absorbent pad AP2, the moving body 4 can be removed from the channel 222. Since the length of the moving substrate 40 is longer than the length of the channel 222, when the moving body 4 moves in the channel 222, only a part of the moving body 4 is accommodated in the channel 222, and the user can move the moving body 4 by gripping the part of the moving body 4 located outside the channel 222 with his / her hand or by clamping with a clamp tool.Since the sample pad 220 is fixed to the connection surface 22b of the bridge portion 22, when the test sample, the first washing liquid, the nucleic acid amplification reagent or the second washing liquid is added, the sample pad 220 is maintained in a fixed position, and after moving the movable body 4 to match the water-absorbing area or the non-water-absorbing area B with the sample pad 220, the test sample, the first washing liquid, the nucleic acid amplification reagent or the second washing liquid can be directly added to the sample pad 220. The present invention can reduce the burden on the user of having to constantly check the current position of the sample pad 220, and achieve the effect of reducing the occurrence of the user adding the test sample, the first washing liquid, the nucleic acid amplification reagent or the second washing liquid in the wrong position.

[0055] Referring to Figures 1, 2, 3, 5, 6 and 11, in each of the following embodiments, the nucleic acid detection device 1 includes a guide body 5, which is a rectangular piece body, one end of the guide body 5 is adhered to one end of the movable body 4, and the multiple openings of the guide body 5 include a first opening O1, a second opening O2 and a third opening O3, and the area directly below the first opening O1 corresponds to the first water absorption region A1 of the movable body 4, the area directly below the second opening O2 corresponds to a part of the non-water absorption region B of the movable body 4, and the area directly below the third opening O3 corresponds to the second water absorption region A2 of the movable body 4. The guide body 5 further includes a plurality of prompt marks, and the bridge portion 22 further includes a guide mark 6. The plurality of prompt marks include a first pattern mark 50, a second pattern mark 51, a third pattern mark 52, a fourth pattern mark 53, a first character mark 54, a second character mark 55, a third character mark 56, a fourth character mark 57, a fifth character mark 58, and a sixth character mark 59. The first pattern mark 50, the second pattern mark 51, the third pattern mark 52, and the fourth pattern mark 53 are all arrow patterns marked by punching on the guide body 5. Among these, the first character mark 54, the second character mark 55, the third character mark 56, the fourth character mark 57, the fifth character mark 58 of the guide body 5, and the sixth character mark 59 of the bridge portion 22 are all prompt characters marked by printing, and the plurality of prompt marks are used to indicate the process sequence corresponding to each opening. The first pattern mark 50 indicates the first opening O1, the second pattern mark 51 indicates the second opening O2, and the fourth pattern mark 53 indicates the third opening O3. Among them, the first opening O1 is marked with the first character mark 54 and the second character mark 55, the second opening O2 is marked with the third character mark 56, and the third opening O3 is marked with the fifth character mark 58. The fourth character mark 57 is marked at a position corresponding to another part of the non-absorbent region B of the moving body 4 of the guide body 5, and the third pattern mark 52 indicates the fourth character mark 57. Here, the sixth character mark 59 is marked on the sample pad 220 on the connection surface 22b of the support 2.Among these, the first character mark 54, the second character mark 55, the third character mark 56, the fourth character mark 57, the fifth character mark 58 and the sixth character mark 59 are represented by text boxes in FIG. 1 and FIG. 2, and the characters to be input into each text box are as follows: the characters of the first character mark 54 are "Step 1: Add test sample", the characters of the second character mark 55 are "Step 2: Add first washing solution", the characters of the third character mark 56 are "Step 3: Add nucleic acid amplification reagent", the characters of the fourth character mark 57 are "Step 4: Wait for nucleic acid amplification reaction", and the characters of the fourth character mark 58 are "Step 5: Wait for nucleic acid amplification reaction". The characters of the fifth character mark 58 are "Step 5: Add second washing liquid and extract mobile body 4", and the characters of the sixth character mark 59 are "Step 6: Add eluent after pressing", the first character mark 54 encourages the execution of the sample addition step S20, the second character mark 55 encourages the execution of the first washing step S25, the third character mark 56 and the fourth character mark 57 encourage the execution of the nucleic acid amplification step S30, the fifth character mark 58 encourages the execution of the second washing step S35, and the sixth character mark 59 encourages the execution of the nucleic acid detection step S40. The user pinches one end of the movable body 4 and the guide body 5 that is bonded thereto, and pushes or pulls the movable body 4 to move the movable body 4 along the direction of the channel 222, or removes the movable body 4 from the channel 222. When performing each process, after the absorbent or non-absorbent area B of the movable body 4 corresponds to the sample pad 220, the user can further press the connection surface 22b with a finger or a clamping tool to sufficiently bring the sample pad 220 on the connection surface 22b into contact with the absorbent area, so as to ensure that the absorbent area can completely absorb the remaining liquid. The above is merely an example, and actual implementation is not limited thereto. In another embodiment of the present invention, the sample pad 220 includes only the nucleic acid adsorption layer 2202, and the nucleic acid extraction layer 2200 is provided in at least one opening corresponding to the water absorption area of ​​the mobile body 4 of the multiple openings. For example, the nucleic acid extraction layer 2200 is provided in the above-mentioned first opening O1, and the nucleic acid extraction layer 2200 can be fixed in the first opening O1 by adhesion, and the upper and lower surfaces of the nucleic acid extraction layer 2200 are exposed. Therefore, when the first opening O1 corresponds to the sample pad 220, the lower surface of the nucleic acid extraction layer 2200 in the first opening O1 contacts the upper surface of the nucleic acid adsorption layer 2202.The above examples are merely illustrative and should not be construed as limiting the scope of the present invention in practice. Any equivalent modifications and variations made to the above content of the present invention should fall within the scope of the present invention.

[0056] Example 1: The operating temperature, operating time and nucleic acid detection range of the nucleic acid detection method provided by the present invention are evaluated using Agrobacterium tumefaciens genomic nucleic acid.

[0057] In Example 1, the genomic nucleic acid of Agrobacterium is used as an example of a test sample, and the virG gene of Agrobacterium is used as an example of a nucleic acid to be detected. In Example 1, a nucleic acid amplification test is performed using the nucleic acid adsorption layer 2202 as an independent component, and a nucleic acid detection test is performed by combining it with the detector 3.

[0058] In the nucleic acid amplification reaction in Example 1, a nucleic acid amplification reagent is added to the nucleic acid adsorption layer 2202 to form an amplicon of the target nucleic acid, and a multi-temperature heating and cooling oscillator is used as a temperature control device during the nucleic acid amplification reaction to control the temperature of the nucleic acid amplification reaction, and a handheld temperature data recorder is used to measure and record the reaction temperature.

[0059] The experimental process of Example 1 is as follows: first, nucleic acid adsorption layer 2202 is placed on a culture dish, and the culture dish is placed on a multi-temperature heating and cooling oscillator; then, a test sample is added to nucleic acid adsorption layer 2202, the nucleic acid in the test sample is adsorbed to nucleic acid adsorption layer 2202, and nucleic acid adsorption layer 2202 is left to stand until it is naturally dried; next, a nucleic acid amplification reagent is added to nucleic acid adsorption layer 2202, and a nucleic acid amplification reaction is performed on the target nucleic acid in nucleic acid adsorption layer 2202 by controlling the temperature of the nucleic acid amplification reaction using a multi-temperature heating and cooling oscillator to form an amplicon of the target nucleic acid; at the same time, during the nucleic acid amplification reaction, the nucleic acid amplification reaction temperature is measured and recorded using a handheld temperature data recorder; and then the nucleic acid amplification reaction temperature is measured and recorded using a handheld temperature data recorder. After the acid amplification reaction is completed, the nucleic acid adsorption layer 2202 is placed in the water absorption area of ​​the mobile body 4 using tweezers, a second washing solution is added to the nucleic acid adsorption layer 2202, the nucleic acid amplification reagent remaining on the nucleic acid adsorption layer 2202 is washed off, and the nucleic acid adsorption layer 2202 is left to stand until it is naturally dried. Next, the nucleic acid adsorption layer 2202 is placed on the initiation pad 30 of the detector 3 using tweezers, and an eluent is added to the nucleic acid adsorption layer 2202. The amplicons of the target nucleic acid in the nucleic acid adsorption layer 2202 are caused to flow together with the eluent to the initiation pad 30, and then from the initiation pad 30 to the binding pad 32, after which they react with the reaction membrane 34, and the amplicons of the target nucleic acid are captured by the detection line 342 of the reaction membrane 34. Hereinafter, the temperature of the nucleic acid amplification reaction is referred to as the "operation temperature", and the time of the nucleic acid amplification reaction is referred to as the "operation time". The test samples each have a copy number of 10 6 , 10 copies 4 , 10 copies 2The Agrobacterium genome nucleic acid with copy number 10, copy number 0, and copy number 0 was tested at operating temperatures of 20°C, 23°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, and the operating times were 5 minutes, 15 minutes, 25 minutes, 35 minutes, 45 minutes, and 55 minutes, respectively. Three repeated experiments were performed for each experimental condition. The detection result was determined to be positive only when both the detection line 342 and the control line 340 on each detection body 3 showed a positive reaction in the three repeated experiments. The positive reaction is, i.e., If colored, it is determined that the detection of the target nucleic acid was successful; if the control line 340 is colored in the detected material in any one of the three repeated experiments and the detection line is not colored, the detection result is determined to be negative; if the result is determined to be positive under the same test conditions, it means that all three repeated experiments are positive reactions, which is represented by "P" and separated by a comma, followed by the numerical value of the minimum copy number of Agrobacterium genomic nucleic acid required for the detection result to be positive; if each group showed a negative result, it is represented by "N", and the experimental results of Example 1 are shown in Table 2 below.

[0060] In the following Table 2, the column represents different working temperatures, and the row represents different working times. For example, the intersection of the column of Table 2 where the column is 5 minutes and the row is 35°C is "P,10 6 The test was repeated three times under the conditions of a working time of 5 minutes and a working temperature of 35°C. In all three tests, the test specimen 3 showed a positive reaction. Therefore, the result was determined to be positive. The minimum number of copies of the Agrobacterium genomic nucleic acid required was 10 6 The test sample is 10 copies. 6 A positive result was obtained only when the Agrobacterium genomic nucleic acid was 10 copies, but 4 Copy, 10 2In addition, for example, the intersection in Table 2 where the column is 15 minutes and the row is 30°C is indicated as "P, 10", and the experiment was repeated three times under the conditions of a working time of 15 minutes and a working temperature of 30°C, and all of the detector 3 showed a positive reaction, so it was determined to be a positive result. The minimum copy number of Agrobacterium genomic nucleic acid required is 10 copies, and a positive result can be obtained when the test sample has 10 copies of Agrobacterium genomic nucleic acid, and a positive result can be obtained when the test sample has 10 copies of Agrobacterium genomic nucleic acid. Therefore, even if the test sample has a higher copy number of Agrobacterium genomic nucleic acid, the result is positive, i.e., 10 6 Copy, 10 4 Copy, 10 2 The test sample was negative only when it had 1 or 0 copies of the Agrobacterium genomic nucleic acid. The above is taken as an example, and the rest should be interpreted similarly.

[0061] The experimental results in Table 2 below show that when the working temperature is 23°C and the working time is 45 minutes, the test sample has a 10% or higher oxidative stress response. 4 A positive result can be obtained only when 10 copies of Agrobacterium genomic nucleic acid are required, that is, all three repeated experiments showed positive reactions. When the working temperature was 23°C and the working time was 55 minutes, the test sample was 10 2 Only a few copies of Agrobacterium genomic nucleic acid are required to detect the target nucleic acid. At the same working temperature, extending the working time helps to detect a smaller number of copies of Agrobacterium genomic nucleic acid, that is, improves the sensitivity of detecting the target nucleic acid. In addition, when the working time is fixed at 55 minutes, Agrobacterium genomic nucleic acid can be detected at working temperatures of 23 to 50°C. When the working temperature is 23°C, the test sample has a copy number of 10 and the target nucleic acid can be detected at a working temperature of 23 to 50°C. 2 The target nucleic acid in the Agrobacterium genomic nucleic acid can be detected, and when the temperature is 25 to 50°C, the test sample only needs to be one copy of the Agrobacterium genomic nucleic acid, and the target nucleic acid can be detected, and the same working time can be achieved. In the above, the sensitivity of detecting the target nucleic acid can be improved with an increase in the working temperature. In addition, the nucleic acid amplification reaction can be accelerated with an increase in the working temperature. For example, when the test sample is one copy of Agrobacterium genomic nucleic acid, a positive result can be observed at a working temperature of 25°C in 45 minutes, but when the working temperature is 30°C, a positive result can be observed in only 25 minutes, when the working temperature is 35°C or 40°C, a positive result can be observed in only 15 minutes, and when the working temperature is 45°C, a positive result can be observed in 5 minutes. The experimental results of Example 1 show that the working temperature at which the amplicon of the target nucleic acid can be obtained is 23 to 50°C, the working time is 5 to 55 minutes, and the target nucleic acid in one copy of Agrobacterium genomic nucleic acid can be detected, so further verification is performed below based on these experimental results. In addition, it should be particularly explained that the above-mentioned working temperature range and working time range do not limit the present invention, and the working temperature range and working time range in the actual implementation are adjusted according to the nucleic acid amplification reagent.

[0062] Table 2: Experimental results of Example 1 [Table 2]

[0063] Example 2: It has been verified that the method of the present invention can be carried out at room temperature using Agrobacterium tumefaciens genome nucleic acid.

[0064] The experimental process of Example 2 is basically the same as that of Example 1, with the only difference being that the nucleic acid amplification reaction is carried out under room temperature environment, so the working temperature is the room temperature when the nucleic acid amplification reaction is carried out, and the working time is fixed at 45 minutes. Three repeated experiments are carried out, and the working temperature is similarly measured and recorded by a handheld temperature data recorder, and the measured maximum and minimum temperatures are taken as the working temperature range. The experimental results are shown in Table 3, which shows the working temperatures measured in each of the three repeated experimental results. The copy number of 10 6 , 10 copies 4 , 10 copies 2 When Agrobacterium genomic nucleic acid with copy number 10, copy number 1, and copy number 0 were used as test samples, the detector 3 was detected to see whether it showed a positive reaction, a positive reaction was indicated by "○" and a negative reaction was indicated by "X", and the group of Agrobacterium genomic nucleic acid with copy number 0 was used as the negative control group. The experimental results showed that when the test sample was Agrobacterium genomic nucleic acid with copy number 10, no positive reaction was obtained in the second test, but positive reactions were obtained in the first and third tests, and the test sample with copy number 10 2In the case of the Agrobacterium genomic nucleic acid, the results of three repeated experiments all showed a positive reaction, i.e., the result was determined to be positive. This result is consistent with the result in Example 1 where the target nucleic acid of the Agrobacterium genomic nucleic acid with a copy number of 1 can be detected when the working temperature is 25°C and the working time is 45 minutes. Therefore, the experimental result in Example 2 shows that the nucleic acid amplification reaction can be completed similarly even in a room temperature environment where the temperature changes, regardless of the control of the working temperature by a temperature control device, and the sensitivity of detecting the target nucleic acid is equivalent to the effect of using a temperature control device. In addition, it is particularly noted that the above-mentioned range of working temperature and range of working time do not limit the present invention, and Example 2 demonstrates that the nucleic acid amplification reaction can be completed without temperature control, and at the same time, Example 1 demonstrates that the nucleic acid amplification reaction time can be shortened with an increase in the working temperature, so in actual implementation, the user may use a household heating device to assist heating, and household heating devices include, for example, a hand warmer or a steam-type eye mask, etc., and for example, during the execution of the nucleic acid amplification step S30, the substrate part 20 is stacked on the hand warmer, and the heat emitted by the hand warmer is transferred to the sample pad 220, thereby improving the speed of the nucleic acid amplification reaction and shortening the required working time. In addition, it is particularly noted that the above-mentioned range of working time and range of working time do not limit the present invention.

[0065] Table 3: Experimental results of Example 2 [Table 3]

[0066] Example 3: Using Agrobacterium, it was verified that the method of the present invention can achieve nucleic acid extraction, amplification and detection of a sample.

[0067] Referring to FIG. 11, the experimental method of Example 3 is based on the nucleic acid detection method provided by the present invention. The nucleic acid detection method includes a first washing step S25 and a second washing step S35, and the steps include a nucleic acid detection device step S10, a sample addition step S20, a first washing step S25, a nucleic acid amplification step S30, a second washing step S35 and a nucleic acid detection step S40, in order. The test sample is an Agrobacterium liquid, and the concentration of Agrobacterium in the Agrobacterium liquid is 100 μL of colony-forming units (CFUs). The Agrobacterium concentration in the Agrobacterium solution in Example 3 is 1.5 x 10 6 CFU / 100μL, 1.5×10 4 CFU / 100μL, 150CFU / 100μL, 15CFU / 100μL, 1.5CFU / 100μL and 0CFU / 100μL. Below, each step of the nucleic acid detection method will be described separately to provide a more specific explanation, and reference will be made to Figures 1, 2, 3, 4, 5, 6, 7 and 11.

[0068] In the step S10 of providing a nucleic acid detection device, the nucleic acid detection device 1 of the present invention is provided, and a specific embodiment of the nucleic acid detection device 1 is as described above.

[0069] In the sample addition step S20, the water absorption area of ​​the moving body 4 is made to correspond to the sample pad 220, and then the test sample is added to the sample pad 220, and the test sample contains the target nucleic acid. In the third embodiment, specifically, the user synchronously moves the moving body 4 and the guide body 5 according to the instruction of the first character mark 54, and the first pattern mark 50 overlaps with the guide mark 6, at this time, the sample pad 220 corresponds to the first opening O1, and at the same time, the sample pad 220 also corresponds to the first water absorption area A1, and since the test sample is an Agrobacterium liquid, after the Agrobacterium liquid is added to the sample pad 220, the Agrobacterium in the Agrobacterium liquid is dissolved by the dissolving liquid in the nucleic acid extraction layer 2200, exposing the nucleic acid of the Agrobacterium, and then the nucleic acid of the Agrobacterium is adsorbed to the nucleic acid adsorption layer 2202, and the remaining liquid is absorbed into the first water absorption area A1.

[0070] In the first cleaning step S25, after the water absorption region of the movable body 4 corresponds to the sample pad 220, a first cleaning liquid is added to the sample pad 220. In the third embodiment, specifically, the user follows the instruction of the second character mark 55 to keep the sample pad 220 in correspondence with the first opening O1 and at the same time corresponds the sample pad 220 to the first water absorption region A1, and then adds the first cleaning liquid to the sample pad 220, causing impurities remaining in the sample pad 220 to flow out together with the first cleaning liquid and be absorbed by the first water absorption region A1.

[0071] In the nucleic acid amplification step S30, the non-water absorbing region B of the mobile body 4 is made to correspond to the sample pad 220, and then the target nucleic acid in the sample pad 220 is reacted with a nucleic acid amplification reagent to form an amplicon of the target nucleic acid. In Example 3, specifically, the user moves the movable body 4 and the guide body 5 synchronously according to the instruction of the third character mark 56, and overlaps the second pattern mark 51 with the guide mark 6. At this time, the sample pad 220 corresponds to the second opening O2. At the same time, the sample pad 220 also corresponds to the non-absorption area B of the movable body 4. Then, add nucleic acid amplification reagent to the sample pad 220. According to the instruction of the fourth character mark 57, the user moves the movable body 4 and the guide body 5 synchronously, and overlaps the third pattern mark 52 with the guide mark 6. At this time, the sample pad 220 is covered by the guide body 5. At the same time, the sample pad 220 also corresponds to the non-absorption area B. Then, according to the instruction of the fourth character mark 57, wait for the progress of the nucleic acid amplification reaction. The working time of the nucleic acid amplification reaction in this embodiment is 45 minutes. The target nucleic acid and the nucleic acid amplification reagent in the sample pad 220 are fully acted on to form an amplicon of the target nucleic acid, and the amplicon of the target nucleic acid is adsorbed by the nucleic acid adsorption layer 2202.

[0072] In the second washing step S35, the absorbent region of the movable body 4 is made to correspond to the sample pad 220, and then the second washing liquid is added to the sample pad 220. In the third embodiment, specifically, the user synchronously moves the movable body 4 and the guide body 5 according to the instruction of the fifth character mark 58, and the fourth pattern mark 53 is overlapped with the guide mark 6, at this time, the sample pad 220 corresponds to the third opening O3, and at the same time, the sample pad 220 also corresponds to the absorbent pad of the second absorption region A2, and then the second washing liquid is added to the sample pad 220, and the nucleic acid amplification reagent remaining in the sample pad 220 is caused to flow out together with the second washing liquid and absorbed in the second region A2.

[0073] In the nucleic acid detection process S40, after the mobile body 4 is removed from the channel 222, the bridge portion 22 is pressed to bend the bridge portion 22, and the sample pad 220 is brought into contact with the initiation pad 30 of the detector 3. Next, an eluent is added to the sample pad 220, and the amplicon of the target nucleic acid in the sample pad 220 is caused to flow to the initiation pad 30 of the detector 3 together with the eluent, and then the amplicon of the target nucleic acid is caused to flow from the initiation pad 30 to at least one control line 340 and at least one detection line 342 of the detector 3, and the amplicon of the target nucleic acid is captured by the at least one detection line 342 mentioned above. In the third embodiment, specifically, after the user pulls out the moving body 4 and the guide body 5, removes the moving body 4 from the channel 222, and separates the guide body 5 from the upper surface of the connection surface 22b of the support 2, the bridge portion 22 can be bent, so that the connection surface 22b of the bridge portion 22 can be pressed with a finger to make the sample pad 220 contact the initiation pad 30, and then the eluent is added to the sample pad 220, and the amplicon of the target nucleic acid in the sample pad 220 flows together with the eluent to the initiation pad 30 of the detection body 3, and then flows from the initiation pad 30 to the binding pad 32, so that the chromogenic substance in the binding pad 32 binds to the amplicon of the target nucleic acid, and the amplicon of the target nucleic acid bound to the chromogenic substance is captured by the detection line 342, forming a visually observable color band, thereby achieving the effect of detecting the target nucleic acid in the test sample, and the unreacted chromogenic substance is captured by the control line 340, and finally the remaining liquid is absorbed by the absorption pad 36.

[0074] The experimental results of Example 3 are shown in Table 4. Table 4 shows the working temperature for each measurement in the experimental results repeated three times, and whether or not the detector 3 showed a positive reaction when the Agrobacterium solution was used as the test sample. A positive reaction is represented by "O", a negative reaction is represented by "X", and the Agrobacterium concentration in the Agrobacterium solution was 1.5 x 10 6 CFU / 100μL, 1.5×10 4CFU / 100μL, 150CFU / 100μL, 15CFU / 100μL, 1.5CFU / 100μL, 0CFU / 100μL, and the group with 0CFU / 100μL of Agrobacterium concentration in the Agrobacterium solution was the negative control group. The experimental results showed that when the Agrobacterium concentration in the Agrobacterium solution of the test sample was 1.5CFU / 100μL, all three repeated experiments showed positive reactions, i.e., positive results were obtained, and in the group with 0CFU / 100μL of Agrobacterium concentration in the Agrobacterium solution, all three repeated experiments showed negative reactions. This means that there were no false positive reactions based on the nucleic acid detection method of the present invention, and using the nucleic acid detection device 1 and nucleic acid detection method of the present invention, the nucleic acid amplification reaction of the target nucleic acid was indeed completed in a room temperature environment without temperature control, an amplicon of the target nucleic acid was obtained, and the amplicon of the target nucleic acid was successfully detected.

[0075] Table 4: Experimental results of Example 3 [Table 4]

[0076] In summary, the nucleic acid detection device and nucleic acid detection method provided by the present invention can indeed complete nucleic acid amplification of a target nucleic acid in a test sample in a room temperature environment without temperature control, obtain an amplicon of the target nucleic acid, and confirm the presence of the target nucleic acid by the amplicon. At the same time, the structure of the nucleic acid detection device provided by the present invention allows a user to easily complete the process from the test sample addition step, the nucleic acid amplification step, to the nucleic acid detection step in a home environment, thereby solving the problem currently lacking in nucleic acid detection devices that can be used at home and have nucleic acid amplification and nucleic acid detection functions.

[0077] Each embodiment is used only to explain the contents of the present invention, and is not intended to limit the scope of the present invention. Therefore, any equivalent changes and modifications based on the claims of the present invention should still be included in the scope of the present invention. [Explanation of symbols]

[0078] 1. Nucleic acid detection device 2. Support 3 Detection object 4. Mobile 5 Guide body 6 Guide Mark 20 Substrate section 22 Bridge section 30 Start Pad 32 Bonding Pad 34 Reaction Membrane 36 Absorbent Pads 40 Moving Board 50 First Pattern Mark 51 Second Pattern Mark 52 3rd Pattern Mark 53 4th Pattern Mark 54 First letter mark 55 Second letter mark 56 Third letter mark 57 4th letter mark 58 5th letter mark 59 6th letter mark 220 Sample Pad 222 Channel 224 Window 340 Control Line 342 Detection Line 2200 Nucleic acid extraction layer 2202 Nucleic acid adsorption layer 22a 1st side wall 22b Connection surface 22c 2nd side wall A1 1st water absorption area A2 2nd water absorption area AA section line AP1 First Absorbent Pad AP2 2nd Absorbent Pad B Non-absorbent area Bu1 First fastening part Bu2 Second fastening part O1 First opening O2 2nd opening O3 3rd opening S10 Nucleic acid detection device provision process S20 Sample addition step S25 First cleaning process S30 Nucleic acid amplification process S35 Second cleaning process S40 Nucleic acid detection process WP Waterproof Pad

Claims

1. The present invention relates to a liquid crystal display device, and a liquid crystal display device, the liquid crystal display device including a support, a detection body, and a moving body, the support including a substrate and a bridge, the bridge being disposed on an upper surface of the substrate, and a channel being formed between the bridge and the substrate, the detection body including a start pad, at least one control line, and at least one detection line, the detection body being disposed on the upper surface of the substrate, the start pad of the detection body being located within the channel, the bridge being provided with a window for viewing the detection result displayed by the at least one control line and the at least one detection line of the detection body, the bridge being provided with a sample pad at a position corresponding to the start pad of the detection body, the moving body being detachably disposed within the channel, and including at least one water absorbing region and at least one non-water absorbing region, A nucleic acid detection device in which the bridge portion and the substrate portion work together to restrict the movement position of the mobile body and move the mobile body along the channel direction, thereby making the absorbent region of the mobile body correspond to the sample pad, or making the non-absorbent region of the mobile body correspond to the sample pad.

2. The nucleic acid detection device according to claim 1 , wherein the sample pad includes a nucleic acid adsorption layer.

3. The nucleic acid detection device according to claim 2 , wherein the sample pad further includes a nucleic acid extraction layer laminated on an upper surface of the nucleic acid adsorption layer.

4. The nucleic acid detection device according to claim 1 , wherein the sample pad further contains a nucleic acid amplification reagent.

5. The nucleic acid detection device of claim 1, further comprising a guide body, one end of the guide body being connected to an upper surface of the movable body, the guide body covering an upper surface of the sample pad of the bridge portion of the support, and a plurality of openings being provided on the guide body, at least one of the plurality of openings corresponding to the water-absorbing region of the movable body, and at least one of the plurality of openings corresponding to the non-water-absorbing region of the movable body.

6. The nucleic acid detection device according to claim 5 , wherein a nucleic acid extraction layer is further provided in at least one of the plurality of openings that corresponds to the water absorption region of the mobile body.

7. a nucleic acid detection device providing step of providing the nucleic acid detection device according to claim 1; a sample addition step of adding a test sample containing a target nucleic acid to the sample pad after the water absorption region of the mobile body is made to correspond to the sample pad; a nucleic acid amplification step of causing the target nucleic acid in the sample pad to react with a nucleic acid amplification reagent after the non-absorbent region of the mobile body corresponds to the sample pad, thereby forming an amplicon of the target nucleic acid; a nucleic acid detection step in which, after removing the mobile body from the channel, the bridge part is pressed to bring the sample pad on the bridge part into contact with the start pad of the detector, an eluent is then added to the sample pad, and the amplicon of the target nucleic acid in the sample pad flows together with the eluent to the start pad of the detector, and then flows from the start pad to at least one control line and at least one detection line of the detector, and the amplicon of the target nucleic acid is captured by the at least one detection line; A method for detecting nucleic acid comprising, in order:

8. The nucleic acid detection method of claim 7, further comprising a first washing step after completion of the sample addition step, in which the water absorption area of ​​the mobile body is aligned with the sample pad and then a first washing liquid is added to the sample pad.

9. The nucleic acid detection method according to claim 7, further comprising a second washing step after completion of the nucleic acid amplification step, in which the water absorption area of ​​the mobile body is aligned with the sample pad and then a second washing liquid is added to the sample pad.

10. 8. The nucleic acid detection method according to claim 7, further comprising a first washing step after completion of the sample addition step, and a second washing step after completion of the nucleic acid amplification step, wherein the first washing step is a step of adding a first washing liquid to the sample pad after corresponding the water absorption area of ​​the mobile body to the sample pad, and the second washing step is a step of adding a second washing liquid to the sample pad after corresponding the water absorption area of ​​the mobile body to the sample pad.

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