High-throughput detection device based on microfluidics and nucleic acid amplification

Abstract

The present invention relates to the field of molecular biology detection, and in particular to a high-throughput detection device based on microfluidics and nucleic acid amplification, comprising: a chip box, including a chip body and a chip cover; the chip body includes a nucleic acid extraction area and a detection area, the nucleic acid extraction area has at least one, and the detection area has two or more; a heating platform, which heats the entire chip box or part of the reaction chamber to reach the reaction temperature of nucleic acid amplification. The detection device of the present invention is easy to operate, and can realize nucleic acid extraction and gene amplification simply and quickly. It has a wide range of uses and is suitable for one-to-many and many-to-many detection needs.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and in particular to a high-throughput detection device based on microfluidics and nucleic acid amplification. Background Art

[0002] With the rapid development of modern biotechnology, the use of molecular detection technology to obtain molecular information in the object to be tested has been widely used. In some cases, in order to obtain test results in a timely and rapid manner, it is necessary to conduct on-site testing of samples. Molecular detection includes three necessary steps: nucleic acid extraction, nucleic acid amplification, and product monitoring. Currently, the commonly used DNA extraction methods in laboratories include CTAB method, SDS method, and kit method. These nucleic acid extraction methods require the use of experimental equipment such as centrifuges and pipettes, and the operation is cumbersome and time-consuming. PCR (polymerase chain reaction) detection technology is a commonly used gene qualitative and quantitative detection technology. For samples with unknown genetic composition, this method requires multiple tests to determine, which is labor-intensive, long cycle, and high cost.

[0003] When multiple tests are performed on the same product, although multiplex PCR can detect several genes at the same time, multiplex PCR in the same reaction tube often produces non-specific amplification and the effect is poor. Complex multiplex PCR primer design also needs to rely on large databases and computer simulation of PCR results, as well as subsequent verification, which places high demands on laboratory instruments and equipment and staff. At present, some institutions and companies at home and abroad have developed gene chip technology for clinical high-throughput testing. However, these detection methods are technically complex, costly, and require expensive instruments to complete. It is impossible to complete the special needs of testing samples containing several, dozens, or dozens of biological genes at one time, especially a large number of biological samples, which requires more effective and rapid high-throughput detection methods. There is an urgent need for a molecular detection device that is simple in structure, easy to operate, and can quickly obtain test results. Summary of the invention

[0004] The present invention provides a high-throughput detection device based on microfluidics and nucleic acid amplification, which combines nucleic acid amplification and microfluidics technology to complete sampling, nucleic acid extraction, amplification and detection at one time, and can simultaneously perform high-throughput detection of multiple genes in one sample; it can also simultaneously perform high-throughput detection of multiple genes in multiple samples.

[0005] The present invention adopts the following technical solution:

[0006] High-throughput detection device based on microfluidics and nucleic acid amplification, including:

[0007] A chip box, comprising a chip body and a chip cover; the chip body comprises a nucleic acid extraction area and a detection area, the nucleic acid extraction area has at least one, and the detection area has two or more;

[0008] A heating platform heats the entire chip box or part of the reaction chamber to reach the reaction temperature of nucleic acid amplification.

[0009] Microfluidics is a technology that precisely manipulates fluids at the micro-nano scale. It can use a small volume of liquid to achieve automatic processing of samples and reagents, rapid response, etc. in a chip, and is very suitable for on-site detection applications. The present invention combines nucleic acid amplification and microfluidics technology, and uses ingenious structural design to integrate the extraction of sample nucleic acid and gene amplification in the same device. The device has a compact structure, small size, small footprint, and is easy to operate. It can easily and quickly achieve nucleic acid extraction and gene amplification. It has a wide range of uses and is suitable for one-to-many and many-to-many detection needs.

[0010] The nucleic acid amplification method can be nucleic acid amplification or variable temperature amplification, and the temperature is controlled by a heating platform.

[0011] Preferably, the nucleic acid extraction area includes a sample chamber, a nucleic acid adsorption chamber and a waste liquid chamber; the sample chamber has an injection hole, and the sample chamber is fluidically connected to the nucleic acid adsorption chamber, the sample chamber is fluidically connected to the waste liquid chamber, and the fluid connection or closing is controlled by a valve; the nucleic acid adsorption chamber has a built-in extraction carrier for nucleic acid molecules to bind.

[0012] Preferably, the detection area comprises at least one reaction chamber and at least one detection chamber; the reaction chamber is in fluid communication with the nucleic acid adsorption chamber, and the reaction chamber is provided with an injection hole.

[0013] Preferably, the chip body further comprises: a cleaning liquid chamber and an elution liquid chamber, both of which are provided with injection holes, and the cleaning liquid chamber and the elution liquid chamber are both in fluid communication with the sample chamber.

[0014] In this solution, the cleaning liquid chamber and the elution liquid chamber are independently set, and the cleaning liquid and the elution liquid are injected separately. The elution liquid or the cleaning liquid can also be pre-stored in the corresponding chamber, and the corresponding valve is closed. There is no need to carry a separate storage device for the eluent and the cleaning liquid, which is more portable. When using, only the corresponding valve needs to be opened. A sealing cover is configured at the opening.

[0015] Preferably, the chip body further comprises: a quantitative cavity, the inlet end of the quantitative cavity is in fluid communication with the sample cavity, and the outlet end is in fluid communication with the reaction cavity. The setting of the quantitative cavity can quantify the reaction solution to meet the quantitative requirements of the detection.

[0016] Preferably, the nucleic acid extraction area is located at the center, and the plurality of detection areas are arranged around the nucleic acid extraction area, forming a circular distribution as a whole.

[0017] Preferably, the chip body is in a foldable fan-shaped structure, and a nucleic acid extraction area and a detection area are distributed on each fan blade.

[0018] Preferably, the chip body is a foldable three-layer structure, with the nucleic acid extraction area arranged on the first layer, the reaction chamber arranged on the second layer, and the detection chamber arranged on the third layer.

[0019] Preferably, the heating platform has a heating slot for all or part of the chip box to be inserted.

[0020] Preferably, the heating platform can be powered by a battery, a power supply or a USB interface, and a solar panel can also be built. The platform is multi-layered and can heat multiple chips at the same time.

[0021] Preferably, the molecular detection device comprises a grinding tool for grinding samples, which is suitable for processing samples that need to be ground, such as fruit, leaf, rhizome and other samples.

[0022] More specifically, the grinding tool includes a grinding rod and a grinding tube, or may only include a grinding rod, and the grinding is performed directly in the sample processing container.

[0023] Preferably, the extraction carrier is filter paper or silica gel membrane.

[0024] Preferably, the detection device further comprises a sampling tool, which comprises a sampling tube and sampling scissors. One end of the sampling tube is a handle for holding by hand, and the other end is cut into an inclined surface. When sampling, the sample to be tested can be scraped and sampled, or the sample to be tested can be pierced into the interior for collection. Depending on the amount of sample required, sampling tubes of different specifications and sizes can be selected. When the sample is hard or dry and is not suitable for the sampling tube, it can be collected with sampling scissors, or the sample can be cut into pieces with sampling scissors to facilitate nucleic acid extraction.

[0025] Preferably, the detection device further comprises an extractor for extracting the lysed sample liquid in the sample processing container into the sample cavity.

[0026] Preferably, the extractor is a syringe with a capillary at the front end.

[0027] When the cleaning liquid or the eluent passes through the sample cavity to clean or elute the sample, the passage of the cleaning liquid or the eluent is blocked due to the presence of the extraction carrier, and the flow speed is very slow. Therefore, it is preferred that a sealing rubber ring is provided at the injection hole of the sample cavity, and the center hole of the sealing rubber ring is adapted to the capillary caliber of the syringe. In this way, after the capillary of the syringe is inserted, a relatively sealed state can be formed between the sealing rubber ring, and the syringe is used to inject into the sample cavity to provide a certain pressure, which is beneficial to the speed of the cleaning liquid or the eluent passing through the sample and improves the cleaning and elution efficiency.

[0028] Similarly, it is also preferred that sealing rubber rings are also provided at other injection holes, such as the injection hole of the reaction chamber and the injection hole of the detection chamber, and the central hole of the sealing rubber ring is adapted to the capillary caliber of the syringe. It is necessary to provide a pushing force for the liquid in the corresponding chamber, and the extractor can be used to push the liquid so that the liquid can enter the next procedure smoothly.

[0029] Preferably, the molecular detection device further comprises a box body for accommodating the sample processing container and the extraction detection box and other components, so as to facilitate the storage, storage and carrying of the entire device. A handle is provided on the box body.

[0030] By implementing the above technical solution, the present invention has the following beneficial effects:

[0031] The present invention combines nucleic acid amplification and microfluidics technology to complete sampling, nucleic acid extraction, amplification, and detection at one time, and can simultaneously perform high-throughput detection of multiple genes in one sample; it can also simultaneously perform high-throughput detection of multiple genes in multiple samples. The detection device of the present invention is a high-throughput detection device with flexible detection methods, simple structural design, and low detection cost, which can realize rapid detection of samples in and results out.

[0032] The detection device of the present invention has a wide range of applications, including but not limited to the following:

[0033] The device and method have a wide range of applications in agriculture, food, medicine, pharmaceuticals, transgenics and other fields. The device and method are not only suitable for the detection of pathogenic microorganisms of crops, pesticide residue detection, and detection of microorganisms in crop production environments such as soil, water, and air; they are also suitable for water source microorganism detection, multiple microorganism detection in a water source sample, and multiple microorganism detection in multiple water source samples; clinical detection and drug screening in medicine; ingredient detection and transgenic detection in food; drug resistance gene screening and resistance gene screening in drug research; and resistance gene screening and high-yield gene screening in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Attached Figure 1 It is a structural schematic diagram of a detection device shown in an embodiment of the present invention;

[0035] Attached Figure 2 It is a schematic structural diagram of a heating platform in a detection device shown in an embodiment of the present invention;

[0036] Attached Figure 3 It is a schematic diagram of the structure of a sample processing container in a detection device shown in one embodiment of the present invention;

[0037] Attached Figure 4 FIG. 1 is a schematic diagram of the structure of an extractor in a detection device shown in an embodiment of the present invention;

[0038] Attached Figure 5 is a schematic structural diagram of a detection device shown in another embodiment of the present invention;

[0039] Attached Figure 6 It is a schematic diagram of the three-dimensional structure of the detection device with a multi-layer structure shown in Example 5 (front view);

[0040] Attached Figure 7 It is a schematic diagram of the three-dimensional structure of the detection device with a multi-layer structure shown in Example 5 (reverse side);

[0041] Attached Figure 8 A schematic diagram of the three-dimensional structure of the multi-layered detection device shown in Example 6 (front view);

[0042] Attached Fig. 9 It is a schematic diagram of the three-dimensional structure of the multi-layer structure detection device shown in Example 6 (back side). DETAILED DESCRIPTION

[0043] The present invention is further described in detail below based on the accompanying drawings and specific embodiments.

[0044] It should be noted that the following implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned implementation cases, those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned implementation cases, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the implementation cases of the present invention.

[0045] Example 1

[0046] This embodiment provides a high-throughput detection device based on microfluidics and nucleic acid amplification, and specifically performs one-to-many qualitative detection using water source microorganism detection as an example.

[0047] The detection device includes a box body, which specifically includes the following structure:

[0048] The sampler can be a pipette, a syringe or other instrument that can sample liquid. The sampler should be used to draw water samples.

[0049] The chip box includes a chip body and a chip cover. The chip cover is made of transparent material, so you can observe any situation on the chip body. The chip body is the part that realizes nucleic acid extraction, reaction, and detection. Specifically, it includes a nucleic acid extraction area and a detection area.

[0050] In this embodiment, there is one nucleic acid extraction area and six detection areas. Figure 1The nucleic acid extraction area is located in the center, and the detection area is arranged around the nucleic acid extraction area, and the overall structure is circular. The chip body can be a corresponding circle or square or other properties, as long as it can accommodate the nucleic acid extraction area and the detection area. The shape of the chip cover is consistent with the shape of the chip body.

[0051] For details, please refer to the attached Figure 1 The nucleic acid extraction area includes: a sample chamber 11, a nucleic acid adsorption chamber 12, a cleaning liquid chamber 13, an elution liquid chamber 14, and a waste liquid chamber 15. The sample chamber 11 has an injection hole 11a, which protrudes from the upper surface of the chip cover to facilitate the injection of samples. The sample chamber 11 is connected to the nucleic acid adsorption chamber 12 through a capillary microfluid, and the nucleic acid adsorption chamber 1 is connected to the waste liquid chamber 15 through a capillary microfluid, and both are controlled to be connected or closed by a capillary valve; the cleaning liquid chamber 13 and the elution liquid chamber 14 are both connected to the sample chamber 11 through a capillary microfluid, and are controlled to be connected or closed by a capillary valve; the nucleic acid adsorption chamber 12 has an extraction carrier for nucleic acid molecules to bind, and the extraction carrier can be a nucleic acid binding filter paper or a silica gel membrane.

[0052] For details, please refer to the attached Figure 1 The detection area includes: a certain volume chamber 19, a reaction chamber 17 and a detection chamber 18, which are connected in sequence through capillaries. The reaction chamber 17 is also provided with an injection hole, and the injection hole of the reaction chamber 17 is used to inject the reaction liquid (nucleic acid amplification reaction liquid). The detection chamber 18 may be provided with an injection hole or not, depending on the detection means. If the detection liquid needs to be used for detection, an injection hole is provided. If the detection is carried out by means of test paper, the test paper is pre-buried in the detection chamber 18, and no additional injection hole is required. A waste liquid chamber connected to the quantitative chamber 19 is also provided. When the liquid reaching the quantitative chamber 19 reaches the target volume, the excess liquid enters the waste liquid chamber.

[0053] The nucleic acid extraction zone is connected to the detection zone via an annular capillary channel. Specifically, the nucleic acid adsorption chamber 12 of the nucleic acid extraction zone is connected to the annular capillary channel, and the quantitative chamber 19 of the detection zone is connected to the annular capillary channel.

[0054] The heating platform 30 has a heating base with a built-in heating wire. The heating part above the heating base has one or more heating grooves. The multiple heating grooves can hold multiple chip boxes to achieve multiple simultaneous heating. The chip box is fully or partially inserted into the heating groove, and the whole or part of the detection area of ​​the chip box is heated to reach the reaction temperature of nucleic acid amplification. Usually, only the reaction chamber 17 of the detection area needs to be heated, but since the chip box is usually small in size, local heating is inconvenient and it is difficult to control the heating position. Therefore, the chip box is heated as a whole.

[0055] This embodiment also provides a detection method, using the above detection device, using a sampler to absorb water samples, injecting the water samples into the sample cavity, flowing into the nucleic acid adsorption cavity, adsorbing the microbial DNA therein, and then injecting cleaning liquid into the cleaning cavity to clean impurities, the cleaning liquid after cleaning flows into the waste liquid cavity, and then injecting elution liquid into the elution liquid cavity to elute DNA as a template for reaction. The eluted liquid with DNA enters the quantitative cavity of each detection area through the annular capillary channel, and after the quantitative is completed, it enters the reaction cavity for nucleic acid amplification reaction. At this time, the chip box is placed on the heating platform and heated at 39°C for 20 minutes. After the reaction is completed, the reaction liquid is introduced into the detection cavity of the detection chip. The detection cavity and the reaction cavity are also one-to-one corresponding. At the same time, the buffer solution is injected, and the detection area is embedded with a test strip. The detection line of the test strip is observed to determine whether the sample contains the gene to be tested. The result can also be determined by adding a color developer and using color changes.

[0056] Example 2

[0057] The detection device of this embodiment is for non-liquid samples such as rice leaves that need to be lysed. In addition to the structure of embodiment 1, the detection device also includes:

[0058] Sampling tools include sampling tubes and sampling scissors. One end of the sampling tube is a handle for holding, and the other end is a bevel. When sampling, the sample can be scraped or inserted into the sample. Depending on the amount of sample required, sampling tubes of different sizes can be selected. When the sample is hard or dry and not suitable for sampling tubes, sampling scissors can be used for collection, or the sample can be cut into pieces with sampling scissors to facilitate nucleic acid extraction.

[0059] The grinding tool is used to grind the sample, and is suitable for processing samples that need to be ground, such as samples such as fruits, leaves, and rhizomes. The grinding tool includes a grinding rod and a grinding tube. The grinding tool is only one implementation method in this embodiment and is not necessary for the molecular detection device of the present invention. For samples that do not need to be ground, a grinding tool is not required, and for samples that need to be ground, it is not necessarily necessary, and the purpose can also be achieved by cutting them into pieces with sampling scissors.

[0060] The sample processing container 20 is a tubular container. Figure 3 As shown, after adding the fragmented sample, add the cell lysis buffer. Some samples do not need to be lysed, so there is no need to add the lysis buffer.

[0061] In the device, except for the chip box which is disposable and cannot be reused, all other components can be reused.

[0062] The extractor 40 extracts the lysed sample liquid in the sample processing container into the sample cavity. Figure 4 As shown, a syringe with a needle at the front end is used, and the needle can be replaced by an adapted capillary, preferably a soft, bendable capillary made of plastic.

[0063] The above detection device is used to perform a one-to-many qualitative detection by taking the early detection of rice blast fungus RAA-LFD as an example. The rice seedling leaves are scraped and sampled with a sampling tube. Fresh rice seedling samples can be directly tested without grinding. The sample is placed in a lysate, 5uL of lysate is drawn after shaking, and the sample is injected into the sample cavity with an extractor, and the sample is controlled to flow through the nucleic acid molecule binding filter paper, DNA is adsorbed in the filter paper, and the remaining liquid enters the waste liquid cavity. Then the cleaning liquid is controlled by the extractor, injected through the injection hole of the sample cavity, and flows through the filter paper for cleaning, and the cleaning liquid enters the waste liquid cavity through the filter paper. Then the extractor is used to inject the eluent through the injection hole of the sample cavity, elute the DNA on the filter paper, and enter the quantitative cavity. After the quantification is completed, it is pushed to the reaction cavity, and the reaction liquid is injected into the reaction cavity through the injection hole, and the reaction liquid can also be sealed in the reaction cavity in advance. The chip box is then heated to 39°C through a heating platform and maintained for 20 minutes to achieve an amplification reaction. After the reaction is completed, the reaction product is pushed to the detection area using an extractor, and a buffer solution is injected from the injection hole. A test strip is embedded in the detection cavity, and the detection line of the test strip is observed to determine whether the sample contains the rice blast gene.

[0064] Embodiment 3:

[0065] This embodiment provides a high-throughput detection device based on microfluidics and nucleic acid amplification, and specifically performs one-to-many qualitative detection using water source microorganism detection as an example.

[0066] In this embodiment, there is one nucleic acid extraction area and four detection areas. The structure of each nucleic acid extraction area and detection area is the same as that in Example 1. The arrangement is different. The detection area of ​​this embodiment is arranged in a fan shape around the nucleic acid extraction area, with multiple fan leaves, as shown in the attached figure. Figure 5 As shown. Each fan blade has a detection area, and the fan leaves can be folded.

[0067] Embodiment 4:

[0068] This embodiment provides a high-throughput detection device based on microfluidics and nucleic acid amplification, and specifically performs many-to-many qualitative detection using water source microorganism detection as an example.

[0069] In this embodiment, four nucleic acid extraction areas and four detection areas are included. The structure of each nucleic acid extraction area and detection area is the same as that in Example 1. The arrangement is different. The four nucleic acid extraction areas of this embodiment are arranged in a row, and the four detection areas are also arranged in a row accordingly.

[0070] Embodiment 5:

[0071] The high-throughput detection device based on microfluidics and nucleic acid amplification provided in this embodiment is a multi-layer structure that realizes one-to-many detection. The multi-layer structure is conducive to reducing the area of ​​the chip body.

[0072] The specific structure is as follows:

[0073] The three-layer structure is similar to the design of a fan and is in a folded state before use, which is convenient for packaging. The layers can be connected by foldable connectors such as paper, or the three layers can be separated from each other. The former makes the detection device more integrated. However, it has limited flexibility in use. The latter has better flexibility in matching, and different numbers of reaction chambers and different numbers of detection chambers can be selected for random matching as needed.

[0074] For the specific structure of this embodiment, please refer to the attached Figure 6 , Attachment Figure 7 .

[0075] The first layer has a substrate, on which a nucleic acid extraction area is arranged, including a sample chamber 11, a nucleic acid adsorption chamber 12, a cleaning liquid chamber 13, an elution liquid chamber 14 and a waste liquid chamber 15. The sample chamber 11 has an injection hole 11a, which protrudes from the upper surface of the chip cover to facilitate the injection of samples. The sample chamber 11 is connected to the nucleic acid adsorption chamber 12 through a capillary microfluid, and the nucleic acid adsorption chamber 1 is connected to the waste liquid chamber 15 through a capillary microfluid, and both are controlled to be connected or closed by a capillary valve; the cleaning liquid chamber 13 and the elution liquid chamber 14 are both connected to the sample chamber 11 through a capillary microfluid, and are controlled to be connected or closed by a capillary valve. Among them, the nucleic acid adsorption chamber 12 can be located on the same side of the substrate, at the bottom, or can be arranged on the other side of the substrate, and the nucleic acid adsorption chamber 12 has a built-in nucleic acid binding filter paper for nucleic acid molecules to bind.

[0076] The second layer also has a substrate, on which is a reaction layer, on which are arranged a reaction chamber 17, a quantitative chamber 19 and a waste liquid chamber 15. The reaction chamber and the quantitative chamber can be provided by one or more, and the same reaction can be performed, and one can be selected for subsequent multiple tests. When multiple different reactions need to be performed, multiple ones can be selected, corresponding to the subsequent multiple over-tests. In this embodiment, three quantitative chambers and three reaction chambers are provided. When elution is performed with an eluent, the outlet of the nucleic acid adsorption chamber 12 is aligned with the quantitative chamber 19 of the second layer. After the quantitative chamber 19 completes the quantification, it enters the reaction chamber 17, and the excess liquid enters the waste liquid chamber. At this time, the first layer is covered on the second layer, and the whole is placed on the heating platform for heating to perform nucleic acid amplification.

[0077] In order to facilitate the elution liquid carrying nucleic acid in the nucleic acid adsorption chamber to enter the quantitative chamber of the reaction layer, a plurality of liquid outlets are provided at the bottom of the nucleic acid adsorption chamber, and a sealing film is sealed on the liquid outlet, and the sealing film can be destroyed under the action of external force. A protrusion with a pointed tip is provided at the injection hole position of the quantitative chamber (located at the center of the injection hole). When the nucleic acid adsorption chamber 12 of the first layer is aligned with the quantitative chamber of the second layer, a little force is applied, and the sealing film at the bottom liquid outlet of the nucleic acid adsorption chamber is punctured by the protrusion with a pointed tip, and at this time, the elution liquid carrying nucleic acid in the nucleic acid adsorption chamber enters the quantitative chamber.

[0078] The third layer also has a substrate, on which is a detection layer, and a detection chamber 18 is arranged. The amplification liquid in the reaction chamber 17 can directly enter the detection chamber 18 through the injection hole of the reaction chamber, and the injection hole of the reaction chamber is aligned with the injection port of the detection chamber 18. However, the amplification liquid is easy to leak out in this way. In this embodiment, it is preferred that an outflow hole is provided on the side wall of the reaction chamber of the reaction layer, and the outflow hole is sealed with a closed film, which can be physically destroyed by external force. Preferably, a protrusion with a pointed tip is provided at the injection hole position (the center position of the injection hole) of the detection chamber of the detection layer. After the nucleic acid amplification is completed, the closed film of the reaction layer is aligned with the injection hole of the detection chamber. At this time, the protrusion with the pointed tip breaks the closed film, and the amplification liquid flows into the detection chamber 18. A detection test paper is pre-buried in the detection chamber 18, and the detection line of the test paper is observed to determine whether the sample contains the gene to be tested.

[0079] See attached Figure 6 , Attachment Figure 7 The three layers of this embodiment are connected by paper material 100. For the convenience of operation, the sample chamber 11, cleaning liquid chamber 13 and elution liquid chamber 14 of the first layer are arranged on one side of the substrate, and the waste liquid chamber 15 and nucleic acid adsorption chamber 12 are arranged on the other side of the substrate. In order to facilitate the alignment of the liquid outlet of the nucleic acid adsorption chamber 12 with the injection port of the quantitative chamber of the second layer and the outlet of the reaction chamber of the second layer with the injection port of the detection chamber of the third layer, from the overall device, the nucleic acid adsorption chamber 12 of the first layer, the reaction chamber of the second layer, and the detection chamber of the third layer are on the same side.

[0080] Embodiment 6:

[0081] The high-throughput detection device based on microfluidics and nucleic acid amplification provided in this embodiment is a multi-layer structure that realizes many-to-many detection. The multi-layer structure is conducive to reducing the area of ​​the chip body.

[0082] The specific structure is as follows:

[0083] The three-layer structure, similar to the fan-shaped design, is in a folded state before use, which is convenient for packaging.

[0084] For the specific structure of this embodiment, please refer to the attached Figure 8 , Attachment Fig. 9 .

[0085] The first layer has a substrate on which nine nucleic acid extraction areas are arranged to achieve nucleic acid extraction of nine samples. The nine nucleic acid extraction areas can include a sample chamber 11, a nucleic acid adsorption chamber 12, a cleaning liquid chamber 13, an elution liquid chamber 14, and a waste liquid chamber 15 for each nucleic acid detection area, or nine sample chambers 11 and nine nucleic acid adsorption chambers 12 are arranged in a nine-square grid, one to one. The cleaning liquid chamber 13, the elution liquid chamber 14, and the waste liquid chamber 15 are shared, and only one is set for each, and the volume of each can be appropriately increased. The latter is adopted in this embodiment. In order to avoid mutual interference between the capillaries connecting the cleaning liquid chamber 13 and the eluting liquid chamber 14 and the waste liquid chamber 15, the connecting pipes of the two are arranged in the upper and lower layers, the capillary pipe network connecting the cleaning liquid chamber 13 and the waste liquid chamber 15 is arranged on the upper layer, and the capillary pipe network connecting the eluting liquid chamber 14 and the waste liquid chamber 15 is arranged on the lower layer, and the sample chamber 11 has an injection hole 11a, which protrudes from the upper surface of the chip cover to facilitate the injection of samples. The sample chamber 11 is connected to the nucleic acid adsorption chamber 12 through a capillary microfluid, and the nucleic acid adsorption chamber 1 is connected to the waste liquid chamber 15 through a capillary microfluid, and both are controlled to be connected or closed by a capillary valve; the cleaning liquid chamber 13 and the eluting liquid chamber 14 are both connected to the sample chamber 11 through a capillary microfluid, and are controlled to be connected or closed by a capillary valve. The nucleic acid adsorption chamber 12 can be located on the same side of the substrate, at the bottom, or on the other side of the substrate, and the nucleic acid adsorption chamber 12 contains a nucleic acid binding filter paper for nucleic acid molecules to bind. The bottom of the nucleic acid adsorption chamber 12 has an outlet, and a closed film is sealed at the outlet. The closed film can be physically destroyed under the action of an external force.

[0086] The second layer has a substrate, on which is a reaction layer, on which nine reaction zones are arranged in a nine-square grid arrangement corresponding to the nucleic acid extraction zone, each reaction zone includes a reaction chamber 17, a quantitative chamber 19 and a waste liquid chamber 15, and there are nine reaction chambers 17 and nine quantitative chambers 19, and the waste liquid chamber 15 shares one, and the volume of the waste liquid chamber 15 is appropriately increased. When using an eluent for elution, align the outlet of the nucleic acid adsorption chamber 12 with the inlet of the quantitative chamber 19 of the second layer, and a protrusion with a pointed tip can be provided at the corresponding position of the inlet of the quantitative chamber 19, and the protrusion with the pointed tip breaks the sealing membrane, and the eluent with nucleic acid enters the quantitative chamber 19, and enters the reaction chamber 17 after the quantitative chamber 19 completes the quantification, and the excess liquid enters the waste liquid chamber. At this time, the first layer is covered on the second layer, and the whole is placed on the heating platform for heating to perform nucleic acid amplification.

[0087] The quantitative chamber 19 and the reaction chamber 17 can be on the same side of the substrate or on different sides, and connected by a capillary in the middle. It depends on the number. When the number is large, arranging them on the same side requires a larger area, which increases the area of ​​the chip body. In this case, it is better to arrange them on different sides.

[0088] The third layer has a substrate, on which is a detection layer, on which nine detection cavities 18 are arranged in a corresponding nine-square grid arrangement. An outflow hole is provided at the bottom of the reaction cavity of the reaction layer, and the outflow hole is sealed with a sealing film, which can be physically destroyed by external force. Preferably, a protrusion with a pointed tip is provided at the injection hole position of the detection cavity of the detection layer (not shown in the figure, please refer to the attached figure). Figure 7 ), when the nucleic acid amplification is completed, the reaction layer is covered on the detection layer, and the bottom of the reaction chamber 17 is aligned with the detection chamber 18. At this time, the protrusion of the tip breaks the sealing membrane, and the amplification liquid flows into the detection chamber 18. A detection test paper is pre-buried in the detection chamber 18. The detection line of the test paper is observed to determine whether the sample contains the gene to be tested.

[0089] See attached Figure 8 , Attachment Fig. 9 In this embodiment, the three layers are independently arranged. In order to facilitate operation and reduce the area of ​​the chip body, the sample chamber 11, the cleaning liquid chamber 13, the elution liquid chamber 14, and the waste liquid chamber 15 of the first layer are located on one side of the substrate, and the nucleic acid adsorption chamber 12 is arranged on the other side of the substrate. The sample chamber 11 and the nucleic acid adsorption chamber 12 are connected through a capillary. The quantitative chamber 19 is located on one side of the second layer substrate, and the reaction chamber 17 is located on the other side of the substrate, and the two are connected through a capillary.

[0090] Embodiment 7:

[0091] This embodiment is different from Embodiment 1 in two aspects:

[0092] 1. Sealing rubber rings are provided at the injection holes of the sample chamber 11, the reaction chamber 17 and the detection chamber 18. The central holes of the sealing rubber rings are adapted to the capillary caliber of the syringe. With this arrangement, after the capillary of the syringe is inserted, a relatively sealed state can be formed between the sealing rubber rings. The syringe is used to inject into the sample chamber 11 to provide a certain pressure, which is conducive to the liquid in the corresponding chamber to smoothly enter the next procedure.

Claims

1. A high-throughput detection device based on microfluidics and nucleic acid amplification, comprising a chip box and a heating platform: The chip box comprises a chip body; The chip body includes a nucleic acid extraction area and a detection area, the nucleic acid extraction area has at least one, and the detection area has two or more; The detection area has at least one reaction cavity and at least one detection chamber, and the reaction cavity is provided with an injection hole; The chip body is a three-layer structure, and the first layer is arranged with a nucleic acid extraction area, including a sample chamber, a nucleic acid adsorption chamber, a cleaning liquid chamber, an elution liquid chamber, and a waste liquid chamber. The sample chamber has an injection hole, and the sample chamber is fluidically connected with the nucleic acid adsorption chamber. The sample chamber is fluidically connected with the waste liquid chamber, and the fluid connection or closing is controlled by a valve; the nucleic acid adsorption chamber is built with a nucleic acid binding filter paper for nucleic acid molecules to bind; the cleaning liquid chamber and the elution liquid chamber are both fluidically connected with the sample chamber; The reaction chamber, the quantitative chamber and the waste liquid chamber are arranged on the second layer, the outlet of the nucleic acid adsorption chamber is aligned with the inlet of the quantitative chamber; the inlet end of the quantitative chamber is in fluid communication with the nucleic acid adsorption chamber, and the outlet end is in fluid communication with the reaction chamber; the reaction chamber is in fluid communication with the quantitative chamber; The detection chamber is arranged on the third layer, and the side wall of the reaction chamber has an outflow hole, and the outflow hole of the reaction chamber is aligned with the injection port of the detection chamber; Each layer is connected by a foldable connector, or the three layers are separated from each other; The heating platform has a heating slot for all or part of the chip box to be inserted into to reach the reaction temperature of nucleic acid amplification.

2. The high-throughput detection device based on microfluidics and nucleic acid amplification according to claim 1, characterized in that: The nucleic acid extraction area is located at a central position, and the plurality of detection areas are arranged around the nucleic acid extraction area.

3. The high-throughput detection device based on microfluidics and nucleic acid amplification according to claim 1, characterized in that: Also includes sampling tools.

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