Centrifugal fully-integrated nucleic acid detection micro-fluidic chip and application thereof

Abstract

The invention discloses a centrifugal fully-integrated nucleic acid detection micro-fluidic chip and application thereof, and belongs to the technical field of nucleic acid detection. The micro-fluidic chip comprises a covering layer and a chip main body which are arranged in a stacked manner, the chip main body comprises a sample cracking area, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative split charging area and a reaction detection area which are communicated through a micro-channel, and the nucleic acid extraction area and the quantitative split charging area are respectively connected with a waste liquid tank through the micro-channel; the sample cracking area, the nucleic acid extraction area, the nucleic acid dilution area, the quantitative split charging area and the waste liquid tank are respectively communicated with the atmosphere through a micro-channel; the reaction detection area is provided with a transparent area; the nucleic acid extraction area comprises a nucleic acid extraction column, and the nucleic acid extraction column comprises an extraction cavity, and a pressing ring and an extraction film which are mounted in the extraction cavity and abut against each other. The micro-fluidic chip disclosed by the invention can realize full-flow integration and accurate control of fluid, is simple and convenient to operate, effectively improves the detection efficiency and the accuracy of a detection result, and can meet the requirements of clinical rapid diagnosis and field detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly relates to a centrifugal fully integrated nucleic acid detection microfluidic chip and its application. Background Art

[0002] Nucleic acid detection is a key technology widely used in modern molecular diagnosis, and plays an important role especially in the fields of infectious diseases, tumor marker detection, and genetic disease screening. Its core steps include sample lysis, nucleic acid extraction, amplification reaction, and detection. The traditional nucleic acid detection process usually requires multiple independent devices and manual operations to complete each link. Although it has high flexibility, it has the following main problems: Sample lysis and nucleic acid extraction require the use of centrifuges, heating devices, and column extraction devices. Nucleic acid amplification requires the use of PCR instruments or isothermal amplification devices. Detection requires the use of fluorescence or colorimetric analyzers. The use of multiple devices not only makes the step process complex and time-consuming, but also makes the detection cost high and requires high professional training for operators, thus greatly limiting the popularization and application of this method. At the same time, the sample transfer between multiple devices and the poor sealing performance of the reaction chambers of existing devices lead to a high risk of contamination and easy introduction of pollution sources, such as aerosol contamination or cross-contamination between samples, affecting the detection specificity and accuracy. In addition, the traditional nucleic acid detection process requires manual handling and dispensing of liquids, which is difficult to ensure the accuracy of liquid quantitative dispensing, affects the sensitivity of subsequent amplification reactions and the accuracy of quantitative analysis, further reduces the result reliability, and is also prone to sample loss or nucleic acid degradation, resulting in low extraction efficiency, insufficient purity, and poor repeatability of experimental results.

[0003] To solve these problems, in recent years, centrifugal microfluidic chips, multi-functional integrated PCR chips, or fully automated portable nucleic acid detection systems have been developed, providing the possibility for the integration and automation of nucleic acid detection. Among them, the centrifugal microfluidic chip integrated with nucleic acid extraction function uses centrifugal force to drive liquid flow, distribution, and processing, designs solid-phase extraction columns or bead adsorption structures in the chip to separate nucleic acids from samples, and realizes automated nucleic acid extraction. However, its sample quantitative dispensing function is weak, and the liquid volume cannot be precisely controlled. At the same time, the amplification reaction and pollution prevention and control designs are relatively simple, with risks of false positives or aerosol contamination. The multi-functional integrated PCR chip performs real-time fluorescence amplification detection by designing a transparent window in the amplification reaction chamber and connecting an external fluorescence detection module, but still requires external equipment such as nucleic acid extractors to cooperate to complete multi-step processes. For example, sample processing and amplification cannot be completed on the same platform, and there is also the problem of aerosol contamination caused by insufficient sealing of the amplification chamber. The fully automated portable nucleic acid detection system completes automated extraction through a kit and pre-loaded reagents, and completes PCR amplification through a built-in small thermal cycling system, thus realizing the integration of nucleic acid extraction and amplification reactions. However, the equipment is complex, and liquid control and sample quantification rely on traditional syringe pumps or external equipment, with high design complexity and maintenance costs, and are not suitable for primary medical institutions or on-site detection.

[0004] In summary, there is an urgent need to develop a centrifugal fully integrated nucleic acid detection microfluidic chip with simple operation to achieve the integration of the whole process of clinical sample nucleic acid detection and precise control of fluids, so as to improve the accuracy and detection efficiency of detection results. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a centrifugal fully integrated nucleic acid detection microfluidic chip that is integrated, efficient, precise, and easy to operate.

[0006] To solve the above problems, the technical solutions adopted by the present invention are as follows: A centrifugal fully integrated nucleic acid detection microfluidic chip, which includes a cover layer and a chip body stacked from top to bottom; the chip body includes a sample lysis area, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative dispensing area, and a reaction detection area that are sequentially connected by microchannels, and the nucleic acid extraction area and the quantitative dispensing area are respectively connected to a waste liquid tank through microchannels; the sample lysis area, the nucleic acid extraction area, the nucleic acid dilution area, the quantitative dispensing area, and the waste liquid tank are respectively communicated with the atmosphere through microchannels; the reaction detection area has a transparent area for fluorescence detection; The nucleic acid extraction area includes a nucleic acid extraction column. The nucleic acid extraction column includes an extraction cavity with a cylindrical structure and a pressing ring hermetically installed in the extraction cavity. A small hole for liquid outflow is formed in the bottom wall of the extraction cavity. An extraction membrane is provided on the bottom wall of the extraction cavity. The pressing ring abuts against the extraction membrane and is used to define the position of the extraction membrane in the liquid flow direction.

[0007] Further preferably, the nucleic acid extraction area further includes a reagent storage pool and a mixing buffer pool. The reagent storage pool and the sample lysis area are respectively connected to the mixing buffer pool through microchannels. The mixing buffer pool is connected to the nucleic acid extraction column. The nucleic acid extraction column is respectively connected to the nucleic acid dilution area and the waste liquid tank through microchannels.

[0008] Further preferably, the reagent storage area includes a precipitant pool, a first cleaning solution pool, a second cleaning solution pool, and an eluent pool. The precipitant pool and the first cleaning solution pool are respectively connected to the mixing buffer pool through microchannels. The eluent pool is connected to the second cleaning solution pool through a microchannel. The second cleaning solution pool is connected to the mixing buffer pool through an inverted U-shaped microchannel.

[0009] As a preferred embodiment of the present invention, the nucleic acid dilution area includes a dilution pool and a diluent storage pool connected to the dilution pool through a zigzag microchannel. The dilution pool is respectively connected to the nucleic acid extraction area and the quantitative dispensing area through microchannels. The microchannel between the dilution pool and the quantitative dispensing area is an S-shaped or serpentine microchannel.

[0010] As a preferred embodiment of the present invention, the quantitative dispensing area includes a liquid channel and a plurality of spaced quantitative slots. The liquid channel is respectively connected to the nucleic acid dilution area and the waste liquid tank through microchannels. Each quantitative slot is respectively connected to the liquid channel.

[0011] As a preferred embodiment of the present invention, a rectifying pool with a downward depression is provided at the connection between the liquid channel and the microchannel connecting the nucleic acid dilution area.

[0012] As a preferred embodiment of the present invention, the reaction detection area includes a plurality of reaction chambers corresponding to the quantitative slots one by one. The reaction chambers are connected to the quantitative slots through microchannels. A paraffin wax tank is provided above the reaction chambers, and paraffin wax is filled in the paraffin wax tank. A blocking groove communicating with the paraffin wax tank is provided on the microchannel between the reaction chamber and the quantitative slot.

[0013] Further preferably, the covering layer is transparent at the position corresponding to the reaction chamber.

[0014] Further preferably, the reaction chamber is pre-filled with LAMP reagent freeze-dried microspheres and fluorescent probes.

[0015] The second object of the present invention aims to provide a nucleic acid detection method based on the above-mentioned centrifugal fully integrated nucleic acid detection microfluidic chip, which comprises the following steps: S1. Pre-place a precipitant, a first cleaning solution, a second cleaning solution and an eluent in the nucleic acid extraction area, pre-place a diluent in the nucleic acid dilution area, and pre-place paraffin, LAMP reagent lyophilized microspheres and a fluorescent probe in the reaction detection area; S2. Add a sample and a lysis solution into the sample lysis area, mix them evenly, and then heat for lysis. After the lysis is completed, a lysis mixture is obtained; centrifuge and rotate the microfluidic chip to make the lysis mixture and the precipitant enter the nucleic acid extraction area and mix evenly for precipitation treatment. Then, centrifuge and rotate the microfluidic chip to make the mixture pass through the nucleic acid extraction column and enter the waste liquid tank; S3. Centrifuge and rotate the microfluidic chip, and the first cleaning solution and the second cleaning solution respectively pass through the nucleic acid extraction column to achieve hierarchical cleaning treatment and then enter the waste liquid tank; S4. Centrifuge and rotate the microfluidic chip to make the eluent elute the captured nucleic acid through the nucleic acid extraction column, and make the eluted nucleic acid enter the nucleic acid dilution area; S5. Centrifuge and rotate the microfluidic chip to mix the nucleic acid and the diluent in the nucleic acid dilution area for dilution; S6. The diluted nucleic acid sample solution to be detected enters the quantitative dispensing area under the drive of centrifugal force, and the nucleic acid sample solution to be detected is quantitatively dispensed into the reaction detection area; S7. The nucleic acid sample solution to be detected enters the reaction detection area and re-dissolves the LAMP reagent lyophilized microspheres. Heat to melt the paraffin, and the molten paraffin plugs the microchannel between the reaction detection area and the quantitative dispensing area under the action of centrifugal force; heat the liquid after re-dissolving and mixing to perform a quantitative amplification reaction and fluorescence signal detection.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The microfluidic chip of the present invention integrates sample lysis, nucleic acid extraction, nucleic acid dilution, quantitative aliquoting, amplification reaction, and detection in the chip body. Through modular design, it realizes full-process integration. Relying on centrifugal force to drive the liquid flow, it does not require an external pump or the cooperation of multiple devices, greatly reducing the intervention of manual operation and the sample transfer time, effectively reducing the risk of sample contamination and loss. It can complete multiple detection steps within the microfluidic chip, reducing the detection cost and operation difficulty. The detection is fast and efficient, greatly improving the repeatability and reliability of the experimental results, and well avoiding the problems such as cumbersome operation, easy contamination, and low precision caused by sample transfer between multiple devices in the traditional nucleic acid detection process. At the same time, the quantitative aliquoting area can ensure the quantitative aliquoting of the sample, making the sample volume in each reaction chamber highly consistent, realizing precise fluid control, and thus significantly improving the sensitivity and quantitative accuracy of the amplification reaction. In addition, by embedding the nucleic acid extraction column into the microfluidic chip and combining it with the microfluidic channel, the automatic flow and separation of the liquid are realized by using centrifugal force, avoiding sample loss and external interference, and ensuring high extraction efficiency and stable nucleic acid purity.

[0017] In summary, the microfluidic chip of the present invention can achieve full-process integration and precise control of the fluid. It is easy to operate, effectively improving the detection efficiency and the accuracy of the detection results, greatly reducing the equipment cost and the operation threshold, and can well meet the needs of clinical rapid diagnosis and on-site detection, especially suitable for popularization and application in resource-limited scenarios such as primary medical institutions. Brief Description of the Drawings

[0018] Figure 1 It is a schematic structural diagram of the chip body of the present invention; Figure 2 It is another schematic structural diagram of the chip body of the present invention; Figure 3 It is a three-dimensional structural diagram of the nucleic acid extraction column of the present invention; Figure 4 It is a sectional view of the nucleic acid extraction column of the present invention; Figure 5 For the present invention Figure 1 Enlarged schematic view of part A; Description of the attached reference numerals: 100, chip body; 110, sample lysis area; 111, sample lysis pool; 120, nucleic acid extraction area; 121, mixing buffer pool; 122, nucleic acid extraction column; 1221, extraction cavity; 1222, pressing ring; 1223, small hole; 1224, extraction membrane; 123, precipitant pool; 124, first cleaning solution pool; 125, second cleaning solution pool; 126, eluent pool; 127, Y-shaped mixing microchannel; 128, inverted U-shaped microchannel; 129, V-shaped diversion groove; 130, nucleic acid dilution area; 131, dilution pool; 132, diluent storage pool; 133, S-shaped or serpentine microchannel; 140, quantitative dispensing area; 141, liquid channel; 142, quantitative groove; 143, rectifying pool; 150, reaction detection area; 151, reaction cavity; 152, paraffin pool; 153, plugging groove; 160, waste liquid pool. Detailed implementation manners

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and the detailed implementation manners.

[0020] As Figures 1 - 2As shown in the figure, the centrifugal fully integrated nucleic acid detection microfluidic chip provided by the present invention includes a cover layer (not shown in the figure) and a chip body 100 which are stacked from top to bottom, and the cover layer is adapted to the chip body 100. Among them, the cover layer can be a cover plate or a cover film, preferably a cover film. Using the cover film to seal the chip body 100 can achieve rapid sealing and low cost, and the cover film is a pressure-sensitive film or a thermosensitive film. Specifically, the chip body 100 includes a sample lysis area 110, a nucleic acid extraction area 120, a nucleic acid dilution area 130, a quantitative dispensing area 140, and a reaction detection area 150 that are sequentially connected through microchannels. The nucleic acid extraction area 120 and the quantitative dispensing area 140 are respectively connected to a waste liquid tank 160 through microchannels; the sample lysis area 110, the nucleic acid extraction area 120, the nucleic acid dilution area 130, the quantitative dispensing area 140, and the waste liquid tank 160 are respectively communicated with the atmosphere through microchannels. The reaction detection area 150 has a transparent area for fluorescence detection. The sample is injected into the sample lysis area 110 for lysis. The lysed sample is washed, captured, and eluted in the nucleic acid extraction area 120. The eluted nucleic acid is diluted in the nucleic acid dilution area 130, and then flows through the quantitative dispensing area 140 to be quantitatively dispensed into the reaction detection area 150 for amplification reaction and fluorescence detection. Among them, the liquid in the nucleic acid extraction area 120 and the excess sample liquid are drained into the waste liquid tank 160 through the microchannel for collection. It can be seen that the present invention integrates sample lysis, nucleic acid extraction and dilution, quantitative dispensing, amplification reaction, and fluorescence detection on the chip body 100 through modular design, connects each area through microchannels, and relies on centrifugal force to drive the liquid flow. The functions are highly integrated, realizing the full-process integration, automation of nucleic acid detection, and precise control of fluid. The operation is simple, and the detection process can be completed without external auxiliary equipment, greatly improving the detection efficiency and the accuracy of the detection results, reducing the equipment procurement and maintenance costs, and also greatly reducing the operation difficulty, which can well meet the high-throughput and rapid detection requirements. Preferably, the chip body 100 is a fan-shaped structure, and the sample lysis area 110, the nucleic acid extraction area 120, the nucleic acid dilution area 130, the quantitative dispensing area 140, and the reaction detection area 150 are sequentially distributed along the radial direction of the chip body 100. The sample injection hole (not shown in the figure) of the sample lysis area 110 is opened on the side wall of the chip body 100 close to the center of the circle, and the sample injection hole can also be set at the position of the cover layer corresponding to the sample lysis area. The sample is directly injected into the sample lysis area through the sample injection hole.

[0021] Specifically, the sample lysis area includes a sample lysis pool 111. The sample and the lysis solution are added to the sample lysis pool 111 through the sample injection hole, and the sample and the lysis solution are mixed under the action of centrifugal force. Uniform heat sources are provided by heating sheets installed on the upper and lower sides of the microfluidic chip for heating and lysis, so as to release nucleic acid. The heating time and temperature during the above lysis process can be precisely regulated by an external control device.

[0022] Specifically, the nucleic acid extraction area 120 includes a reagent storage pool, a mixing buffer pool 121, and a nucleic acid extraction column 122 that are sequentially arranged from top to bottom, thereby ensuring that the flow path of the reagent under the action of centrifugal force is unidirectional flow, avoiding cross-contamination. The reagent storage pool and the sample lysis area are respectively connected to the mixing buffer pool 121 through microchannels, the mixing buffer pool 121 is connected to the nucleic acid extraction column 122, and the nucleic acid extraction column 122 is respectively connected to the nucleic acid dilution area 130 and the waste liquid tank 160 through microchannels. Further, the reagent storage area specifically includes a precipitant pool 123, a first cleaning solution pool 124, a second cleaning solution pool 125, and an eluent pool 126. The precipitant pool 123 and the first cleaning solution pool 124 are respectively connected to the mixing buffer pool 121 through microchannels, wherein the precipitant pool 123 is located below the first cleaning solution pool 124 and is arranged with the same diameter as the sample lysis area, and the length of the microchannel at the outlet of the first cleaning solution pool 124 is greater than the length of the microchannel at the outlet of the sedimentation pool; preferably, the microchannel at the outlet of the first cleaning solution pool 124 has a certain arc. The microchannel at the outlet of the precipitant pool 123 is connected to the microchannel between the sample lysis area 110 and the nucleic acid extraction area 120 to form a Y-shaped mixing microchannel 127, which can further improve the mixing effect between the lysis mixture and the precipitant. The eluent pool 126 is connected to the second cleaning solution pool 125 through a microchannel, and the second cleaning solution pool 125 is connected to the mixing buffer pool 121 through an inverted U-shaped microchannel 128. The second cleaning solution flows unidirectionally through the inverted U-shaped microchannel 128 under the action of centrifugal force and enters the mixing buffer pool 121. The inverted U-shaped microchannel 128 can ensure that the flow path length of the second cleaning solution is greater than the flow path length of the first cleaning solution, effectively ensuring the cleaning order of the first cleaning solution and the second cleaning solution, and at the same time, the inverted U-shaped microchannel 128 can effectively prevent liquid backflow. Similarly, the eluent in the eluent pool 126 needs to pass through the second cleaning solution pool 125, the inverted U-shaped microchannel 128, and the mixing buffer pool 121 in sequence to flow into the nucleic acid extraction column 122 to elute the nucleic acid, ensuring the orderly progress of cleaning and elution. It can be seen that the present invention makes the order of steps such as precipitation, cleaning, and elution through a special liquid flow path design.

[0023] More specifically, the nucleic acid extraction column 122 includes an extraction cavity 1221 in a cylindrical structure and a pressing ring 1222 hermetically installed in the extraction cavity 1221. A small hole 1223 communicating with the extraction cavity 1221 is formed in the bottom wall of the extraction cavity 1221 for allowing liquid to flow out. An extraction membrane 1224 is disposed on the bottom wall of the extraction cavity 1221, and the extraction membrane 1224 is preferably a silica gel membrane. The pressing ring 1222 abuts against the extraction membrane 1224 and is used to define the position of the extraction membrane 1224 in the liquid flow direction, ensuring the stability of the extraction membrane 1224 and the controlled fluid flow path. The sample liquid enters the nucleic acid extraction column 122 by centrifugal force. The first cleaning liquid and the second cleaning liquid sequentially flow through the nucleic acid extraction column 122 and enter the waste liquid tank 160 under the action of centrifugal force, realizing hierarchical cleaning, effectively removing impurities in the sample liquid. The extraction membrane 1224 can efficiently adsorb nucleic acid. After the cleaning is completed, the eluent flows through the extraction column under the action of centrifugal force, and the purified nucleic acid is eluted into the downstream nucleic acid dilution area 130. Further, the microchannels between the nucleic acid extraction column 122 and the waste liquid tank 160 and the nucleic acid dilution area 130 are in an inverted Y-shaped structure, and the liquid flow direction can be controlled by controlling the centrifugal direction. Specifically, the cleaning liquid enters the waste liquid tank 160 by rotating the microfluidic chip counterclockwise, and the eluted nucleic acid enters the nucleic acid dilution area 130 by rotating the microfluidic chip clockwise. In the present invention, by embedding the nucleic acid extraction column 122 into the microfluidic chip and combining it with the microfluidic channel, the automatic flow and separation of liquid are realized by using centrifugal force, avoiding sample loss and external interference, and ensuring high extraction efficiency and stable nucleic acid purity. In the present invention, the extraction cavity 1221 of the nucleic acid extraction column 122 can be integrally formed in the chip body 100, which can effectively reduce the manufacturing cost, or can be installed in the chip body 100 as an independent component by an assembly method, making it easy to install the extraction membrane 1224 and the pressing ring 1222. In some embodiments, such as Figure 3 and Figure 4 shown, the nucleic acid extraction column 122 is in a block structure, and an extraction cavity 1221 is provided inside. A small hole 1223 with an inner diameter smaller than that of the extraction cavity 1221 is formed in the bottom wall of the extraction cavity 1221. The small hole 1223 communicates with the extraction cavity 1221 and penetrates through the nucleic acid extraction column 122. The extraction membrane 1224 is disposed at the connection between the small hole 1223 and the extraction cavity 1221. The pressing ring 1222 is installed in the extraction cavity 1221 and is in interference fit with the extraction cavity 1221. The pressing ring 1222 abuts against the extraction membrane 1224 to limit it; correspondingly, such as Figure 5As shown in the figure, a receiving cavity matching the nucleic acid extraction column 122 is provided inside the chip body 100. This receiving cavity is located below the mixing buffer pool 121, and the nucleic acid extraction column 122 is installed in this receiving cavity. To ensure that the liquid in the extraction cavity 1221 can be quickly discharged, the inner diameter of the small hole 1223 cannot be too small, which makes it difficult to match the width of the microchannel. Therefore, a V-shaped diversion groove 129 is provided below the receiving cavity. The bottom wall and both side walls of the V-shaped diversion groove 129 are inclined surfaces, which can better play the role of confluence and diversion.

[0024] Specifically, the nucleic acid dilution area 130 includes a dilution pool 131 and a diluent storage pool 132 connected to the dilution pool 131 through a zigzag microchannel. The dilution pool 131 and the diluent storage pool 132 are respectively connected to the atmosphere through microchannels. The dilution pool 131 is arranged on one side of the mixing buffer pool 121 and is slightly lower than the mixing buffer pool 121. The dilution pool 131 is connected to the nucleic acid extraction area 120 and the quantitative dispensing area 140 through microchannels respectively. The microchannel between the dilution pool 131 and the quantitative dispensing area 140 is an S-shaped or serpentine microchannel 133. The diluent enters the dilution pool 131 under the action of centrifugal force to dilute the eluted nucleic acid, and the diluted nucleic acid sample solution to be tested enters the quantitative dispensing area 140 under the drive of centrifugal force.

[0025] Specifically, the quantitative dispensing area 140 includes a liquid channel 141 and a plurality of equally sized and spaced quantitative slots 142. The liquid channel 141 is connected to the nucleic acid dilution area 130 and the waste liquid tank 160 through microchannels respectively. Each quantitative slot 142 is connected to the liquid channel 141 respectively. The nucleic acid sample solution to be tested is driven into a plurality of equally sized quantitative slots 142 by centrifugal force, realizing accurate quantitative dispensing of the sample, ensuring the quantitative consistency of the sample amplification reaction, effectively guaranteeing the sensitivity, accuracy and reliability of the subsequent amplification reaction, and well avoiding the problem of quantitative error caused by manual operation or complex equipment in the prior art, which affects the amplification result. To further ensure the quantitative accuracy of sample dispensing, a rectifying pool 143 with a downward depression is provided at the connection between the liquid channel 141 and the microchannel connecting the nucleic acid dilution area 130. The rectifying pool 143 can buffer the sample solution entering the quantitative dispensing area 140, avoiding the problem of inaccurate quantification in the first reaction cavity 151 due to the flow rate and flow deviation of the sample solution.

[0026] Specifically, as Figure 5As shown in the figure, the reaction detection area 150 includes a plurality of reaction chambers 151 arranged in one-to-one correspondence with the quantitative tanks 142. The reaction chambers 151 are communicated with the quantitative tanks 142 through microchannels, and the sample enters the reaction chambers 151 from the quantitative tanks 142. Each reaction chamber 151 is pre-loaded with freeze-dried microspheres of LAMP reagent (loop-mediated isothermal amplification reagent) and fluorescent probes. After the sample enters the reaction chamber 151, the freeze-dried microspheres of LAMP reagent can be quickly and automatically re-dissolved to ensure the reaction sensitivity and specificity. Above the reaction chamber 151, there is a paraffin tank 152 filled with paraffin. On the microchannel between the reaction chamber 151 and the quantitative tank 142, there is a blocking groove 153 communicated with the paraffin tank 152. The present invention adopts the paraffin blocking technology. Before the amplification reaction, the paraffin is melted by heating, and then the microchannel at the entrance of the reaction chamber 151 is blocked by centrifugal force to form a sealing layer, physically isolating the aerosol pollution source, effectively avoiding aerosol leakage and cross-contamination between samples during the amplification process, thereby reducing the risk of false positives in the results, significantly improving the detection specificity, safety and accuracy, and being particularly suitable for the multi-sample high-throughput detection scenario. Of course, the blocking agent is not limited to paraffin, and other commonly used blocking agents in the art can also be used. In order to improve the sealing performance of the paraffin to the entrance of the reaction chamber 151, the blocking groove 153 is preferably in a V-shaped structure, and the microchannel between the blocking groove 153 and the paraffin tank 152 is inclined, so that the paraffin can quickly seal the blocking groove 153 under the action of centrifugal force. The present invention adopts the LAMP isothermal amplification technology instead of the traditional PCR amplification technology, realizing low-cost and high-efficiency nucleic acid amplification under constant temperature conditions in the microfluidic chip, significantly reducing the equipment cost and energy consumption. Further, the bottom wall of the reaction chamber 151 is transparent, and correspondingly, the covering layer corresponding to the position of the reaction chamber 151 is also transparent to form a transparent detection window. After the amplification reaction is completed, an external fluorescence detection module is used to collect real-time fluorescence signal data through the transparent detection window, and the amplification curve is analyzed and displayed through an external Android all-in-one machine.

[0027] In summary, through modular design, the microfluidic chip of the present invention integrates sample lysis, nucleic acid extraction, nucleic acid dilution, quantitative dispensing, amplification reaction and detection into the chip main body 100, realizing full-process integration. Through the embedding of the nucleic acid extraction column 122, the LAMP isothermal amplification technology and the paraffin blocking technology, as well as the precise control of the quantitative dispensing area 140, the problems of low detection efficiency, high sample pollution risk, poor fluid control accuracy and complex operation existing in the prior art are solved.

[0028] The nucleic acid detection method of the above centrifugal fully integrated nucleic acid detection microfluidic chip specifically includes the following steps: S1. Pre-set a precipitant, a first cleaning solution, a second cleaning solution, and an elution solution in the nucleic acid extraction area 120, pre-set a dilution solution in the nucleic acid dilution area 130, and pre-set paraffin, LAMP reagent freeze-dried microspheres, and fluorescent probes in the reaction detection area 150; S2. Add the sample and the lysis solution to the sample lysis area and mix them evenly, then heat (for example, heat at 56 °C for 10 min) for lysis to release nucleic acids. After lysis, a lysis mixture is obtained; centrifuge and rotate the microfluidic chip to make the lysis mixture and the precipitant enter the nucleic acid extraction area 120 and mix evenly (for example, the mixing time is 10 s) for precipitation treatment, and make the mixture pass through the nucleic acid extraction column 122 by centrifuging and rotating the microfluidic chip and then enter the waste liquid tank 160; S3. Centrifuge and rotate the microfluidic chip, and the first cleaning solution and the second cleaning solution respectively pass through the nucleic acid extraction column 122 to achieve hierarchical cleaning treatment and then enter the waste liquid tank 160; S4. Centrifuge and rotate the microfluidic chip to elute the captured nucleic acids with the elution solution through the nucleic acid extraction column 122, and make the eluted nucleic acids enter the nucleic acid dilution area 130; S5. Centrifuge and rotate the microfluidic chip to mix the nucleic acids and the dilution solution in the nucleic acid dilution area 130 for dilution; S6. The diluted nucleic acid sample solution to be tested enters the quantitative dispensing area 140 under the drive of centrifugal force, and the nucleic acid sample solution to be tested is quantitatively dispensed into the reaction detection area 150; S7. The nucleic acid sample solution to be tested enters the reaction detection area 150 and re-dissolves the LAMP reagent freeze-dried microspheres. Heat to melt the paraffin, and the molten paraffin plugs the microchannel between the reaction detection area 150 and the quantitative dispensing area 140 under the action of centrifugal force; heat the liquid after re-dissolving and mixing to perform a quantitative amplification reaction and fluorescence signal detection. Example

[0029] A nucleic acid detection method for bovine mastitis pathogens based on a centrifugal fully integrated nucleic acid detection microfluidic chip is as follows: I. Reagents 1. DNA extraction reagents The DNA extraction reagents include a lysis solution, proteinase K, a first cleaning solution, a second cleaning solution, and an elution solution. Among them, the components of the lysis solution include guanidine hydrochloride, Tween-20, Triton, SDS, and EDTA; the components of the first cleaning solution include guanidine hydrochloride, NaCl, Tris-HCl, and isopropanol; the components of the second cleaning solution include Tris-HCl and absolute ethanol; the components of the elution solution include Tris-HCl and EDTA.

[0030] 2. Kit for detecting six pathogens of bovine mastitis by LAMP isothermal amplification method The LAMP reaction solution contains LAMP buffer, Bst DNA polymerase, MgSO4, fluorescent dye, mannitol, trehalose, bovine serum albumin, PEG20000, and 5-6 LAMP primers each capable of specifically detecting Staphylococcus aureus ( Staphylococcus aureus ), Streptococcus agalactiae ( Streptococcus agalactiae ), Streptococcus uberis ( Streptococcus uberi s), Mycoplasma bovis ( Mycoplasmopsis bovis ), Klebsiella ( Klebsiella spp. ), and Streptococcus dysgalactiae ( Streptococcus dysgalactiae ). The specific LAMP primer sequences for each pathogen are shown in Table 1.

[0031] The above reaction system can be prepared in the form of freeze-dried microspheres for pre-placement in the reaction chambers of a microfluidic chip. Different reaction chambers can be pre-placed with LAMP reaction freeze-dried spheres containing primers for different pathogens, and the function of the microfluidic chip can be utilized to achieve the function of simultaneously detecting six pathogens.

[0032] Table 1 List of LAMP primer sequences for six pathogens related to bovine mastitis

[0033] II. Sensitivity test for nucleic acid detection of bovine mastitis pathogens based on the microfluidic chip of the present invention To test the sensitivity of nucleic acid detection of six bovine mastitis pathogens on the microfluidic chip of the present invention, plasmids of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Mycoplasma bovis, Klebsiella, and Streptococcus dysgalactiae were mixed into a milk sample so that the concentration of each plasmid in the sample reached 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL. Samples containing different concentrations of plasmids were added to the microfluidic chip, and the detection of six targets was carried out according to the operation steps of the following detection method: 1. Experimental preparation 1.1 Sample preparation The plasmids of the above six pathogens were respectively mixed into a sterile milk sample to prepare test samples with different concentration gradients. The concentration of each plasmid was 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10³ copies / mL, and 10² copies / mL. Three parallel samples were prepared for each concentration sample to ensure the repeatability of the results.

[0034] 1.2 Reagent Pre - setting Pre - set a precipitant (such as ethanol or isopropanol, with a concentration of 70% - 100% and a volume of 50 - 200 μL), a first washing solution (such as a buffer salt solution containing 70% - 80% ethanol, with a volume of 100 - 300 μL), a second washing solution (such as a low - salt buffer solution containing 50% - 70% ethanol, with a volume of 100 - 300 μL), and an elution solution (such as deionized water or a low - salt TE buffer solution, with a volume of 20 - 100 μL) in the nucleic acid extraction area.

[0035] Pre - set a dilution solution (such as nuclease - free water or a dilution buffer solution, with a volume of 50 - 200 μL) in the nucleic acid dilution area.

[0036] Pre - set paraffin (with a melting point of 50 - 60°C and a volume of 10 - 50 μL), LAMP reagent freeze - dried microspheres (containing specific primers, DNA polymerase, and reaction buffer salts, designed for six pathogens), and a fluorescent probe (such as a SYTO - 9 or FAM - labeled probe, with a concentration of 0.1 - 1 μM) in the reaction detection area.

[0037] 2. Detection Steps 2.1 Sample Lysis Add 200 - 500 μL of milk samples containing different concentrations of plasmids and 500 - 1000 μL of lysis solution (such as a buffer solution containing proteinase K and surfactant) to the sample lysis area, and mix well. Heat at 55 - 65°C for 5 - 15 minutes for lysis to obtain a lysis mixture. Place the microfluidic chip in a centrifuge and centrifuge at a speed of 500 - 2000 rpm for 1 - 3 minutes to allow the lysis mixture and the precipitant to enter the nucleic acid extraction area and mix evenly. Let it stand for 1 - 5 minutes for nucleic acid precipitation. Centrifuge again (1000 - 3000 rpm, 1 - 3 minutes) to allow the mixture to pass through the nucleic acid extraction column, and the waste liquid flows into the waste liquid tank.

[0038] 2.2 Nucleic Acid Washing Centrifuge the microfluidic chip (1000 - 3000 rpm, 1 - 2 minutes) to allow the nucleic acid after precipitation treatment to be mixed with the first washing solution and the second washing solution in sequence for hierarchical washing. After each washing, centrifuge (1000 - 3000 rpm, 1 - 2 minutes) to allow the waste liquid to flow through the nucleic acid extraction column into the waste liquid tank to complete the purification of the nucleic acid.

[0039] 2.3 Nucleic Acid Elution Centrifuge the microfluidic chip (1000 - 3000 rpm, 1 - 2 minutes) to allow the elution solution to be mixed with the washed nucleic acid, and elute the captured nucleic acid through the nucleic acid extraction column. The eluted nucleic acid enters the nucleic acid dilution area under the action of centrifugal force.

[0040] 2.4 Nucleic Acid Dilution Centrifuge the microfluidic chip (500 - 2000 rpm, 1 - 2 minutes) to fully mix the eluted nucleic acid with the diluent in the nucleic acid dilution area, obtaining the diluted nucleic acid sample solution to be tested.

[0041] 2.5 Quantitative Sub - packaging Centrifuge the microfluidic chip (1000 - 3000 rpm, 1 - 3 minutes) to enable the diluted nucleic acid sample solution to enter the quantitative sub - packaging area under the drive of centrifugal force. Through the microchannel design in the quantitative sub - packaging area, the nucleic acid sample solution to be tested is evenly sub - packaged into multiple reaction and detection areas (the sample volume in each reaction area is 5 - 25 μL).

[0042] 2.6 LAMP Amplification and Detection After the nucleic acid sample solution to be tested enters the reaction and detection area, it contacts and redissolves with the LAMP reagent dry microspheres. Heat at 60 - 65 °C for 1 - 5 minutes to melt the paraffin. The molten paraffin plugs the microchannel between the reaction and detection area and the quantitative sub - packaging area under the action of centrifugal force (500 - 2000 rpm) to prevent the reaction solution from flowing back or cross - contaminating. Continue to heat at 60 - 65 °C for 30 - 60 minutes to carry out the LAMP quantitative amplification reaction, and simultaneously monitor the fluorescence signal in real - time through a fluorescence detection device (excitation wavelength 470 - 495 nm, emission wavelength 510 - 530 nm).

[0043] The detection results are shown in Table 2 Table 2 Summary Table of the Detection Sensitivity Results of the Kit

[0044] From the results in Table 2, it can be known that the LAMP method detection sensitivity of the six pathogens of bovine mastitis using the microfluidic chip of the present invention can reach 1000 copies / mL, indicating that the DNA extraction efficiency and detection efficiency of the microfluidic chip of the present invention for bovine mastitis samples can reach a relatively high level.

[0045] III. Detection of Clinical Samples of Bovine Mastitis Pathogens Collect bovine mastitis samples positive for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Mycoplasma bovis, Klebsiella, and Streptococcus dysgalactiae (Ct values between 28 - 33 detected by qPCR), add them to the microfluidic chip for the whole - process detection from DNA extraction to LAMP amplification, and the detection results are shown in Table 3.

[0046] Table 3 Detection Results of Clinical Bovine Mastitis Positive Samples

[0047] From the results in Table 3, it can be seen that by using the microfluidic chip of the present invention in combination with the LAMP bovine mastitis pathogen detection reagent, bovine mastitis-related pathogens can be effectively detected, and the detection results are consistent with those of qPCR.

[0048] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection required by the present invention.

Claims

1. A centrifugal fully integrated nucleic acid detection microfluidic chip, characterized in that: The invention comprises a covering layer and a chip body stacked from top to bottom; the chip body comprises a sample lysis area, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative sub-packaging area and a reaction detection area connected through a microfluidic channel, the nucleic acid extraction area and the quantitative sub-packaging area are respectively connected to a waste liquid tank through a microfluidic channel; the sample lysis area, the nucleic acid extraction area, the nucleic acid dilution area, the quantitative sub-packaging area and the waste liquid tank are respectively connected to the atmosphere through the microfluidic channel; the reaction detection area has a transparent area for fluorescence detection; The nucleic acid extraction area includes a nucleic acid extraction column, which includes an extraction cavity with a cylindrical structure and a pressure ring sealed in the extraction cavity. A small hole for liquid to flow out is opened on the bottom wall of the extraction cavity. An extraction membrane is arranged on the bottom wall of the extraction cavity. The pressure ring is against the extraction membrane and is used to limit the position of the extraction membrane in the flow direction of the liquid.

2. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The nucleic acid extraction area also includes a reagent storage pool and a mixing buffer pool. The reagent storage pool and the sample lysis area are respectively connected to the mixing buffer pool through microfluidics. The mixing buffer pool is connected to the nucleic acid extraction column. The nucleic acid extraction column is respectively connected to the nucleic acid dilution area and the waste liquid tank through microfluidics.

3. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The reagent storage area includes a precipitant tank, a first cleaning liquid tank, a second cleaning liquid tank and an eluent tank. The precipitant tank and the first cleaning liquid tank are connected to a mixing buffer tank through microchannels, respectively. The eluent tank is connected to the second cleaning liquid tank through a microchannel. The second cleaning liquid tank is connected to the mixing buffer tank through an inverted U-shaped microchannel.

4. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The nucleic acid dilution area includes a dilution pool and a dilution liquid storage pool connected to the dilution pool through a zigzag microchannel. The dilution pool is connected to the nucleic acid extraction area and the quantitative filling area through microchannels respectively. The microchannel between the dilution pool and the quantitative filling area is an S-shaped or serpentine microchannel.

5. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that: The quantitative subpackaging area includes a liquid channel and a plurality of quantitative grooves arranged at intervals. The liquid channel is connected to the nucleic acid dilution area and the waste liquid groove through microfluidics, and each of the quantitative grooves is connected to the liquid channel.

6. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 5, characterized in that: A downwardly recessed rectifying pool is provided at the connection between the liquid channel and the microchannel connected to the nucleic acid dilution zone.

7. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 5, characterized in that: The reaction detection area includes a plurality of reaction chambers arranged in one-to-one correspondence with the quantitative grooves, and the reaction chambers are connected to the quantitative grooves through microchannels; a paraffin groove is arranged above the reaction chamber, and the paraffin groove is filled with paraffin; a blocking groove connected to the paraffin groove is arranged on the microchannel between the reaction chamber and the quantitative groove.

8. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 7, characterized in that: The position of the covering layer corresponding to the reaction chamber is transparent.

9. The centrifugal fully integrated nucleic acid detection microfluidic chip according to claim 7, characterized in that: The reaction chamber is pre-installed with LAMP reagent freeze-dried microspheres and fluorescent probes.

10. A nucleic acid detection method based on the centrifugal fully integrated nucleic acid detection microfluidic chip according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1, pre-pour a precipitant, a first cleaning solution, a second cleaning solution and an eluent into the nucleic acid extraction area, pre-pour a diluent into the nucleic acid dilution area, and pre-pour paraffin, LAMP reagent freeze-dried microspheres and a fluorescent probe into the reaction detection area; S2, adding the sample and the lysis solution into the sample lysis zone and mixing them evenly, heating them for lysis, and obtaining a lysis mixture after the lysis is completed; centrifuging the microfluidic chip to allow the lysis mixture and the precipitant to enter the nucleic acid extraction zone and mix them evenly for precipitation treatment, and centrifuging the microfluidic chip to allow the mixture to pass through the nucleic acid extraction column and enter the waste liquid tank; S3, centrifugally rotating the microfluidic chip, and the first cleaning solution and the second cleaning solution respectively pass through the nucleic acid extraction column to achieve graded cleaning treatment and then enter the waste liquid tank; S4, centrifugally rotating the microfluidic chip, allowing the eluent to elute the captured nucleic acid through the nucleic acid extraction column, and allowing the eluted nucleic acid to enter the nucleic acid dilution zone; S5, centrifugally rotating the microfluidic chip to mix the nucleic acid and the diluent in the nucleic acid dilution area for dilution; S6. The diluted nucleic acid sample solution to be tested enters the quantitative dispensing area under the driving force of centrifugal force, so that the nucleic acid sample solution to be tested is quantitatively dispensed into the reaction detection area; S7. The nucleic acid sample liquid to be tested enters the reaction detection area and the LAMP reagent freeze-dried microspheres are re-dissolved, and the paraffin is heated to melt. The molten paraffin blocks the microchannel between the reaction detection area and the quantitative filling area under the action of centrifugal force; the re-dissolved and mixed liquid is heated to perform quantitative amplification reaction and fluorescence signal detection.

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