Nucleic acid extraction chip, bacterial microfluidic integrated detection platform and detection method

Abstract

The application provides a nucleic acid extraction chip, a bacterial micro-fluidic integrated detection platform and a detection method. The nucleic acid extraction chip comprises a shunt layer, a filter layer and a flow aggregation layer which are closely attached in sequence; a sample inlet of the shunt layer is connected with a plurality of shunt ports through a plurality of first micro-channels; the filter layer comprises filter paper for enriching nucleic acid; a sample outlet of the flow aggregation layer is connected with a plurality of flow aggregation ports through a plurality of second micro-channels, and the number and distribution of the flow aggregation ports correspond to those of the shunt ports; the filter paper is arranged between the flow aggregation ports and the shunt ports. The detection platform comprises a bacterial lysis unit and a nucleic acid amplification reaction unit; the bacterial lysis unit comprises the nucleic acid extraction chip and a first temperature control module. The nucleic acid extraction chip and the detection platform have less demand for samples and reagents, shorter time consumption, reduced infection risk of detection personnel and improved extraction efficiency. The isothermal amplification method is adopted, the sensitivity is high, the reagent is pre-frozen and embedded in the detection hole, and the operation process is simplified.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a nucleic acid extraction chip, a bacterial microfluidic integrated detection platform, and a detection method. Background Technology

[0002] Bacterial diseases remain a serious threat to human health. Traditional bacterial identification, requiring three steps—enrichment, selective culture, and biochemical identification—is considered the "gold standard" for bacterial detection. However, its drawbacks include its long processing time (3-5 days) and low sensitivity, making it unsuitable for rapid and accurate responses to disease outbreaks and failing to meet the requirements for on-site, real-time testing. With the continuous development of molecular biology techniques, numerous detection technologies established based on the study of biological macromolecules, particularly nucleic acids, at the molecular level have become highly anticipated new products and are gradually being applied to the rapid identification of bacteria.

[0003] Nucleic acid extraction is fundamental to biological reactions and is crucial for the accuracy and timeliness of downstream amplification. While the commonly used column method can obtain DNA of a certain purity, it requires multiple centrifugations, demands large sample volumes, is unsuitable for automation, and poses potential health hazards to operators. Microfluidic chips offer advantages such as small size, low reagent consumption, high throughput, and short processing time, showing promise in nucleic acid detection. Furthermore, in nucleic acid amplification, recombinase polymerase amplification (RPA) is an isothermal amplification technique that can be performed at room temperature (37–42 °C) with a reaction time of only 10–20 minutes, making it suitable for on-site detection and showing a trend of replacing PCR.

[0004] In summary, there is a need to design a nucleic acid extraction chip and a microfluidic platform that integrates nucleic acid extraction and detection to solve the problems of cumbersome processes, high time and labor costs in current bacterial detection technologies. Summary of the Invention

[0005] In view of the shortcomings of the existing technology in bacterial detection, such as cumbersome process, high time and labor costs, the purpose of this invention is to provide a nucleic acid extraction chip that requires less sample and reagents, takes less time, reduces the risk of infection for testing personnel, and improves extraction efficiency.

[0006] The nucleic acid extraction chip provided by this invention includes a flow divider layer, a filter layer, and a flow aggregation layer that are tightly fitted together from top to bottom. The flow divider layer has an inlet port, which is connected to multiple flow dividers through multiple first microchannels. The filter layer includes filter paper for enriching nucleic acids. The flow aggregation layer has an outlet port, which is connected to multiple flow aggregation ports through multiple second microchannels. The number and distribution of the flow aggregation ports correspond to the flow dividers. The inlet port, the first microchannels, the flow dividers, the flow aggregation ports, the second microchannels, and the outlet port are connected in sequence, and the filter paper is disposed between the flow aggregation ports and the flow dividers.

[0007] By designing the nucleic acid extraction chip with a multi-layered structure, the vertical space is fully utilized, which facilitates chip miniaturization. The sample entering from the inlet is diverted and diffused to multiple outlets, effectively expanding the processing space and area for sample heating, lysis, purification, and elution, thereby significantly improving nucleic acid extraction efficiency. Compared to traditional bacterial nucleic acid extraction methods, the nucleic acid extraction chip microfluidic integrated platform provided by this invention requires less sample and reagents, consumes less time, reduces the risk of infection for testing personnel, and improves extraction efficiency.

[0008] Preferably, the filter layer further includes two support layers, on which multiple support units are provided. The number and distribution of the support units correspond to the diversion port and the convergence port. Each support unit is provided with multiple support columns and through holes at intervals. The through holes are used to connect the convergence port or the diversion port. The support units on the two support layers are arranged opposite to each other and clamp the filter paper.

[0009] Preferably, the filter paper is glass fiber filter paper or Whatman No.1 filter paper with a pore size of 0.5~1.0μm. Filter paper with a pore size range can ensure extraction effect while taking into account extraction rate.

[0010] The present invention also provides a bacterial microfluidic integrated detection platform, including a bacterial lysis unit and a nucleic acid amplification reaction unit; the bacterial lysis unit includes a nucleic acid extraction chip and a first temperature control module, the first temperature control module being used to control the temperature of the nucleic acid extraction chip; the nucleic acid amplification reaction unit is used to perform isothermal amplification and detection of the nucleic acid extracted by the bacterial lysis unit.

[0011] This microfluidic integrated platform requires less sample and reagent, consumes less time, reduces the risk of infection for testing personnel, and improves extraction efficiency. It employs an isothermal amplification method, eliminating the need for nucleic acid denaturation, resulting in high sensitivity. Furthermore, pre-lyophilized reagents embedded in the reaction wells simplify the operational process.

[0012] Preferably, the nucleic acid amplification reaction unit includes a sample detection chip and a second temperature control module. The second temperature control module is used to control the temperature of the sample detection chip, and the sample detection chip is used to amplify and detect the nucleic acid extracted by the bacterial lysis unit at an isothermal temperature.

[0013] Preferably, the sample detection chip has an injection port, which is connected to at least one pair of parallel reaction detection ports and positive / negative control ports via a third microchannel. The reaction detection ports are pre-embedded with RPA nucleic acid amplification reagent, which includes RPA buffer, specific primers for the target bacterial gene, and fluorescent probes.

[0014] Preferably, the RPA nucleic acid amplification reagent is freeze-dried at -80°C and then encapsulated in the reaction detection well.

[0015] Preferably, it further includes a housing and a signal reading unit; the signal reading unit includes a fluorescence excitation light source and an image acquisition device; the fluorescence excitation light source and the nucleic acid amplification reaction unit are disposed inside the housing, and the image acquisition device is used to acquire fluorescence signals during the isothermal amplification and inspection process of nucleic acid.

[0016] Preferably, the angle between the light beam emitted by the fluorescent excitation source and the sample detection chip is 7~9°.

[0017] Preferably, the device further includes a first filter and a second filter. The first filter is disposed between the fluorescent excitation light source and the nucleic acid amplification reaction unit, and the transmission band of the first filter is the blue light band. The second filter is disposed between the nucleic acid amplification reaction unit and the image acquisition device, and the transmission band of the second filter is the green light band.

[0018] Preferably, the housing includes a lower housing and an upper housing; the lower housing is used to house the bacterial lysis unit and the nucleic acid amplification reaction unit; the upper housing is fastened to the lower housing; the fluorescence excitation light source is disposed inside the upper housing and located above and to the side of the nucleic acid amplification reaction unit; a through hole is also provided on the upper housing, the through hole being located directly above the nucleic acid amplification reaction unit; the image acquisition device is disposed outside the housing and acquires the fluorescence intensity during the isothermal amplification and inspection process of nucleic acid through the through hole.

[0019] Preferably, the lower housing is provided with a first loading module, a second loading module, a liquid guide pipe, and a waste liquid collection device;

[0020] The first loading module includes a first mounting slot with an upper opening and a second mounting slot with a side opening; the first mounting slot is used to mount the nucleic acid extraction chip, and a drain port is provided at the bottom of the first mounting slot; the second mounting slot is used to mount the first temperature control module.

[0021] The second loading module is disposed to the side and below the first loading module. The second loading module includes a third mounting slot with an upper opening and a fourth mounting slot with a side opening. The third mounting slot is used to install the sample detection chip. The fourth mounting slot is used to install the second temperature control module.

[0022] One end of the liquid guide tube is connected to the drain port, and the other end is connected to the waste liquid collection device or the sample detection chip.

[0023] Preferably, both the first temperature control module and the second temperature control module include a silicone rubber heating plate and a heating controller connected to each other.

[0024] The present invention also provides a bacterial detection method, which uses the bacterial microfluidic integrated detection platform described above for detection. First, the bacterial culture medium is injected into the nucleic acid extraction chip, and after heating, lysis, purification and elution, nucleic acid is obtained. The obtained nucleic acid is introduced into the nucleic acid amplification reaction unit for isothermal amplification and detection.

[0025] Preferably, lysis is performed by isothermal heating at 70~100℃ for at least 30 min; isothermal amplification and detection are performed by isothermal heating at 37~42℃ for 10~20 min.

[0026] Compared with existing technologies, the nucleic acid extraction chip, bacterial microfluidic integrated detection platform and detection method provided by this invention require less sample and reagent, consume less time, reduce the risk of infection for testing personnel, and improve extraction efficiency.

[0027] (1) Compared with traditional bacterial nucleic acid extraction methods, this microfluidic integrated platform requires less sample and reagent, takes less time, reduces the risk of infection for testing personnel, and improves extraction efficiency.

[0028] (2) The isothermal amplification method is adopted, which does not require nucleic acid denaturation step, has high sensitivity, and the reagents are pre-ly lyophilized and embedded in the reaction wells, which simplifies the operation process.

[0029] (3) When combined with intelligent equipment, it is easier to promote and use the microfluidic integrated platform, which is suitable for fields such as food safety, clinical testing, and environmental monitoring.

[0030] The above-mentioned technical features can be combined in various suitable ways or replaced by equivalent technical features, as long as the purpose of the present invention can be achieved. Attached Figure Description

[0031] The invention will now be described in more detail based on embodiments that are merely non-limiting and with reference to the accompanying drawings. Wherein:

[0032] Figure 1 This is an exploded view of a nucleic acid extraction chip provided in an embodiment of the present invention;

[0033] Figure 2 An exploded view of a sample detection chip provided in an embodiment of the present invention;

[0034] Figure 3 An exploded view of a bacterial microfluidic integrated detection platform provided in an embodiment of the present invention;

[0035] Figure 4 An exploded view of the housing provided in an embodiment of the present invention;

[0036] Figure 5 This is a schematic diagram of the inner side of the lower housing according to an embodiment of the present invention;

[0037] Figure 6 This is a schematic diagram of the optical path of a bacterial microfluidic integrated detection platform provided in an embodiment of the present invention.

[0038] Explanation of reference numerals in the attached figures:

[0039] 100. Nucleic acid extraction chip; 101. Inlet; 102. First microchannel; 103. Splitting port; 104. Converging port; 105. Second microchannel; 106. Outlet; 107. Filter paper; 108. Support unit; 109. Support column; 110. Through hole; 200. Sample detection chip; 201. Inlet port; 202. Third microchannel; 203. Reaction detection well; 204. Positive and negative control well; 205. Substrate layer; 300. First temperature control module; 400. Second temperature control module; 500. LED excitation light source; 600. Image acquisition device; 700. Lower shell; 701, First mounting slot; 702, Second mounting slot; 703, Third mounting slot; 704, Fourth mounting slot; 705, Drain outlet; 706, Waste liquid collection device; 800, Upper housing; 801, Slot; 802, Through hole; 901, First filter; 902, Second filter. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Based on the specific embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] The terms "first," "second," and similar words used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Words such as "including" or "contains" mean that the element preceding the word encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as "above," "below," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.

[0042] In this disclosure, when a specific device is described as being located between a first device and a second device, an intermediary device may or may not be present between the specific device and the first or second device. When a specific device is described as being connected to other devices, the specific device may be directly connected to the other devices without an intermediary device, or it may be not directly connected to the other devices but have an intermediary device.

[0043] All terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in a general dictionary, such as a dictionary, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or highly formalized meaning, unless expressly defined herein.

[0044] like Figure 1 As shown, the nucleic acid extraction chip 100 includes a diversion layer, a filter layer, and a convergence layer that are tightly attached from top to bottom.

[0045] A sample inlet 101 is located at the center of the flow divider. Multiple flow dividers 103 and multiple first microchannels 102 are evenly distributed around the sample inlet 101, with the number of flow dividers 103 and first microchannels 102 being the same. In this embodiment, four flow dividers 103 and four first microchannels 102 are provided, with the four first microchannels 102 connecting the four flow dividers 103 to the sample inlet 101 respectively. The diameter of the sample inlet 101 is preferably 1-5 mm, and the width-to-depth ratio of the first microchannels 102 is preferably 1:6.

[0046] A sample outlet 106 is located at the center of the flow-gathering layer. Multiple flow-gathering ports 104 and multiple second microchannels 105 are evenly distributed around the sample outlet 106. The number of flow-gathering ports 104 and second microchannels 105 is the same as the number of flow-diverting ports 103. In this embodiment, four second microchannels 105 connect the four flow-gathering ports 104 to the sample outlet 106, respectively. The distribution positions of the four flow-gathering ports 104 on the flow-gathering layer correspond to the distribution positions of the flow-diverting ports 103 on the flow-diverting layer. The diameter of the sample outlet 106 is preferably 1~5 mm, and the width-to-depth ratio of the second microchannels 105 is preferably 1:6.

[0047] The filter layer includes upper and lower support layers and filter paper 107 sandwiched between the two support layers. The upper and lower support layers are permanently bonded by plasma oxidation treatment. The two support layers have the same structure, and each support layer is provided with multiple support units 108. The number of support units 108 is consistent with the number of diversion ports 103. In this embodiment, four support units 108 are provided, and the distribution positions of the four support units 108 correspond to the distribution positions of the diversion ports 103 and the convergence ports 104. Each support unit 108 is provided with multiple support pillars 109 and through holes 110 at intervals. The support units 108 on the two support layers are arranged opposite each other, and the support pillars 109 on the two support layers cooperate to clamp the filter paper 107, which is used to enrich nucleic acids. By providing support pillars 109 to support the filter paper 107, it is beneficial to form a pressure difference across the filter paper 107, thereby improving the filtration efficiency. For the upper support layer, its support unit 108 is positioned perpendicular to the top diversion port 103 and has a larger area than the corresponding diversion port 103. Its through-hole 110 is connected to the diversion port 103. For the lower support layer, its support unit 108 is positioned perpendicular to the bottom convergence port 104 and has a larger area than the corresponding convergence port 104. Its through-hole 110 is connected to the convergence port 104. The diversion layer, filter layer, and convergence layer are tightly fitted together, allowing the sample inlet 101 to sequentially connect to the sample outlet 106 via the first microchannel 102, diversion port 103, filter paper 107, convergence port 104, and second microchannel 105, forming a channel from the sample inlet 101 to the sample outlet 106. By diverting and diffusing the sample entering from the sample inlet 101 to multiple diversion ports 103, the processing space and area for sample heating, lysis, purification, and elution are effectively expanded, thereby effectively improving the nucleic acid extraction efficiency. Compared to traditional bacterial nucleic acid extraction methods, the nucleic acid extraction chip 100 microfluidic integrated platform provided by this invention requires less sample and reagent, takes less time, reduces the risk of infection for testing personnel, and improves extraction efficiency.

[0048] In the above embodiments, the filter paper 107 is preferably nucleic acid enrichment filter paper 107, and more preferably glass fiber filter paper or Whatman No. 1 filter paper. The diameter of the filter paper 107 is preferably 3-15 mm, and the pore size is preferably 0.5-1.0 μm; more preferably, the diameter of the filter paper 107 is preferably 5-9 mm, and the pore size is preferably 0.7-0.9 μm. The height of the support column 109 is preferably 50-500 μm, and the diameter is preferably 0.5-5 mm; more preferably, the height of the support column 109 is preferably 100-300 μm, and the diameter is preferably 1-3 mm.

[0049] The nucleic acid extraction chip 100 provided by this invention can be applied to a bacterial microfluidic integrated detection platform.

[0050] like Figure 3 As shown, the bacterial microfluidic integrated detection platform provided by this invention mainly includes a bacterial lysis unit and a nucleic acid amplification reaction unit. The bacterial lysis unit is used to extract nucleic acids from bacteria, and the nucleic acid amplification reaction unit is used to amplify and detect the nucleic acids extracted by the bacterial lysis unit at an isothermal temperature.

[0051] The bacterial lysis unit includes a first temperature control module 300 and a nucleic acid extraction chip 100 provided by the present invention. The first temperature control module 300 is used to control the temperature of the nucleic acid extraction chip 100. The nucleic acid amplification reaction unit includes a sample detection chip 200 and a second temperature control module 400. The second temperature control module 400 is used to control the temperature of the sample detection chip 200, which is used for isothermal amplification and detection of the nucleic acid extracted from the bacterial lysis unit.

[0052] like Figure 2 As shown, the sample detection chip 200 includes a top layer and a base layer 205 bonded together. A sample inlet 201 is located at the center of the top layer. Multiple third microchannels 202 and multiple pairs of parallel reaction detection wells 203 and positive / negative control wells 204 are evenly distributed around the sample inlet 201. The reaction detection wells 203 are pre-embedded with RPA nucleic acid amplification reagent, which includes RPA buffer, specific primers for the target bacterial gene, and fluorescent probes. Preferably, the RPA nucleic acid amplification reagent is freeze-dried at -80°C to form a lyophilized agent, which is then encapsulated in the reaction detection wells 203. The number of reaction detection wells 203 and positive / negative control wells 204 is determined by the number and types of pathogens to be detected. On the sample detection chip 200, preferably, the injection port 201, the third microchannel 202, the reaction detection port 203, and the positive and negative control ports 204 are distributed in a centrally symmetrical pattern. The diameter of the injection port 201 is preferably 1~5 mm, the width-to-depth ratio of the third microchannel 202 is preferably 2:10, and the diameters of the reaction detection port 203 and the positive and negative control ports 204 are preferably 1~8 mm.

[0053] Both the first temperature control module 300 and the second temperature control module 400 include a connected silicone rubber heating plate and a heating controller, with a preferred heating power of 0.01-2 W / cm². 2 .

[0054] To facilitate the reading of test results, the bacterial microfluidic integrated detection platform is also equipped with a signal reading unit. To facilitate the assembly of various components, protect the parts, and shield against external light to ensure accurate signal reading, a housing can also be added. For example... Figure 4 , Figure 5As shown, the housing includes a lower housing 700 and an upper housing 800 that can be interlocked. The lower housing 700 is used to house the bacterial lysis unit and the nucleic acid amplification reaction unit. The signal reading unit includes a fluorescence excitation light source and an image acquisition device 600; wherein the fluorescence excitation light source is located inside the upper housing 800, and the light emitted by it illuminates the sample detection chip 200, and the image acquisition device 600 is used to acquire fluorescence signals during the isothermal amplification and inspection process of nucleic acid.

[0055] Specifically, the lower housing 700 is provided with a first loading module, a second loading module, a liquid guide pipe, and a waste liquid collection device 706.

[0056] The first loading module is used to load the bacterial lysis unit. It includes a first mounting slot 701 with an upper opening and a second mounting slot 702 with a side opening. The first mounting slot 701 is used to mount the nucleic acid extraction chip 100. Its width and length are greater than the nucleic acid extraction chip 100, and its depth is preferably 10-15 mm. A drain port 705 is also provided at the center of the bottom of the first mounting slot 701. The drain port 705 is used for the passage of a liquid guide tube (not shown in the figure), and the diameter of the drain port 705 is preferably 1-2 mm. The second mounting slot 702 is used to mount the silicone rubber heating plate of the first temperature control module 300.

[0057] The second loading module includes a third mounting slot 703 with an upper opening and a fourth mounting slot 704 with a side opening. The third mounting slot 703 is used to mount the sample detection chip 200, and its width and length are greater than the sample detection chip 200, with a depth preferably of 5-10 mm. The fourth mounting slot 704 is used to mount the silicone rubber heating plate of the second temperature control module 400. Preferably, the second loading module is located to the side and below the first loading module so that the fluid discharged from the sample extraction chip can enter the sample detection chip 200 under the action of gravity.

[0058] A waste liquid collection device 706 is provided between the first loading module and the second loading module. In some embodiments, the waste liquid collection device 706 includes a centrifuge tube placement tank, and centrifuge tubes are added for waste liquid collection.

[0059] One end of the liquid guide tube is connected to the drain port 705, and the other end can be selectively connected to the waste liquid collection device 706 or the sample inlet 201 of the sample detection chip 200.

[0060] The upper housing 800 has a slot 801 for mounting a fluorescent excitation light source. When the upper housing 800 is fastened to the lower housing 700, the fluorescent excitation light source is located above and to the side of the nucleic acid amplification reaction unit. The fluorescent excitation light source is preferably an LED excitation light source 500. The fluorescent excitation light source needs to form a surface light source on the detection chip plane. Considering the scattering of the light source, to improve the accuracy of signal reading, the angle between the beam emitted by the fluorescent excitation light source and the sample detection chip 200 is preferably 5~10°, more preferably 7~9°. The fluorescent excitation light source forms a 2~4 mm light spot on the plane of the sample detection chip 200. The upper housing 800 also has a through hole 802. When the upper housing 800 is fastened to the lower housing 700, the through hole 802 is located directly above the nucleic acid amplification reaction unit. The distance between the through hole 802 and the sample detection chip 200 is preferably 5~8 cm, more preferably 6~7 cm. An image acquisition device 600 is located outside the housing and captures images of the sample detection chip 200 through a through-hole 802, thereby acquiring fluorescence signals during the isothermal amplification and examination of nucleic acid. The image acquisition device 600 can be a mobile phone, tablet, or camcorder capable of video recording. The recorded video is processed using self-developed programs in C, R, Python, or other languages.

[0061] To improve the accuracy of signal reading, a first filter 901 is placed between the fluorescence excitation source and the sample detection chip 200, and a second filter 902 is placed between the sample detection chip 200 and the image acquisition device 600. Both filters are dichroic mirrors. The transmission band of the first filter 901 is the blue light band, more preferably 480nm±5nm, and the transmission band of the second filter 902 is the green light band, more preferably 520nm±5nm.

[0062] This invention also provides a bacterial detection method, which uses the bacterial microfluidic integrated detection platform provided in this application for detection, and the detection steps include:

[0063] First, bacterial culture medium is injected into nucleic acid extraction chip 100. After heating, lysis, purification, and elution, nucleic acid is obtained. The obtained nucleic acid is then introduced into the nucleic acid amplification reaction unit for isothermal amplification and detection.

[0064] Heating lysis is performed at a high temperature of 70-100℃, preferably by constant temperature heating for at least 30 min. After lysis, the DNA product is washed with purification buffer and elution buffer sequentially to obtain a high-quality DNA product. Nucleic acid amplification refers to the RPA amplification reaction, in which specific primers and fluorescent probes for bacterial target genes are added to the reaction system and reacted at 37-42℃ for 10-20 min.

[0065] The fluorescence signal reading during the isothermal amplification and testing of nucleic acid is performed using an image acquisition device 600 (e.g., using a mobile phone to record video). The video recording of the fluorescence signal is then processed using self-written programs in C, R, Python, etc.

[0066] Example 1: Preparation of Nucleic Acid Extraction Chip 100

[0067] A flow distribution layer and a flow collection layer with channel structures, as well as upper and lower support layers with four circular support units 108, were prepared using a masking method. The flow distribution layer, the two support layers, and the flow collection layer were all made of PMDS. The inlet 101 of the flow distribution layer and the outlet 106 of the flow collection layer each have a diameter of 1 mm. The flow distribution port 103 and the flow collection port 104 each have a diameter of 4 mm. The first microchannel 102 and the second microchannel 105 each have a length of 12 mm and a width and depth of 100 μm. The circular support unit 108 has a diameter of 10 mm, and each support unit 108 contains 31 cylindrical support pillars 109 with a height of 200 μm and a diameter of 1 mm. A 2 mm hole 110 is punched in the support unit 108 to connect the filter layer to the upper diversion port 103 and the lower convergence port 104. A glass fiber filter paper 107 with a diameter of 8 mm and a pore size of 0.8 μm is sandwiched between the support pillars 109 of the upper and lower support layers for bacterial filtration and nucleic acid sample extraction. The diversion layer, two support layers, and convergence layer are stacked sequentially, and adjacent layers are permanently bonded together through plasma oxidation treatment. The overall chip size is 5×5×3 cm. 3 Range. The preferred plasma oxidation treatment conditions are 300 mT and 20 W for 60 s.

[0068] Example 2: Fabrication of Sample Detection Chip 200

[0069] A sample inlet 201, multiple third microchannels 202, multiple pairs of reaction detection wells 203, and positive / negative control wells 204 are formed on the top layer. The sample inlet 201 is located in the center of the top layer, and the reaction detection wells 203 and positive / negative control wells 204 are distributed around the sample inlet 201 and connected to it through the third microchannels 202. Finally, the top layer is bonded to the glass substrate 205. The diameter of the sample inlet is 1 mm, and the diameters of the reaction detection wells 203 and positive / negative control wells 204 are 5 mm. The third microchannel 202 is 100 μm wide and 200 μm deep. The reaction detection wells 203 are pre-loaded with RPA reagent containing specific primers and fluorescent probes for common foodborne pathogens, and then covered with a transparent sealing film to prevent evaporation during the reaction process. The overall chip size is 2 × 2 × 1 cm. 3 scope.

[0070] Example 3: Construction of the Detection Platform

[0071] like Figure 3 As shown, the shell is 3D printed. The bacterial lysis unit and nucleic acid amplification reaction unit are mounted inside the lower shell 700, and the signal reading unit is mounted on the upper shell 800. The bacterial lysis unit is located on the left side of the lower shell 700, and the nucleic acid extraction chip 100 is located at a size of 6×6×10 cm. 3 The first mounting slot 701 has a 2 mm drain outlet 705 in the center. The amplification reaction unit is located on the right side of the lower housing 700, with the sample detection chip 200 located in a 3×3×5 cm space. 3 The third mounting slot 703 is located within the housing. Additionally, a centrifuge tube placement slot is provided for waste liquid collection. The top of the upper housing 800 has a 1.5 × 2 cm... 2 The through-hole 802 serves as the imaging port, and contains a light source slot 801 for housing the LED excitation light source 500. The overall dimensions of the detection platform are 16 × 10 × 7 cm. 3 scope.

[0072] The optical path of the detection platform is as follows Figure 6 As shown, the arrows indicate the direction of light propagation. A first filter 901 is placed between the LED excitation light source 500 and the sample detection chip 200. Preferably, the first filter 901 is in close contact with the focusing ring of the LED excitation light source 500 to filter out stray light from outside the excitation light source. The scattering angle of the light source is 8°, forming a 3mm light spot on the sample detection chip 200. A second filter 902 is placed between the sample detection chip 200 and the through-hole 802 to filter out stray light from outside the detection light source. The image acquisition device 600 is an iPhone 12, and the distance between it and the chip is 6.5 cm.

[0073] Example 4: Bacterial Detection Case

[0074] The detection targets include eight common foodborne pathogens: Salmonella, Vibrio parahaemolyticus, Escherichia coli, Cronobacter sakazakii, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, and Bacillus cereus. Detection principle: In the RPA reaction system, when the target gene is present, it can be specifically cleaved by exonucleases, leading to the generation of a fluorescent signal, thereby enabling quantitative analysis of the bacteria.

[0075] The nucleic acid extraction chip 100 was prepared as described in Example 1. The sample detection chip 200 was prepared as described in Example 2, with lyophilized reagents of the above-mentioned eight pathogen-specific primers and fluorescent probes pre-embedded in the reaction detection wells 203, and then sealed with a light-transmitting sealing film for preservation. The detection device was set up as described in Example 3, and the nucleic acid extraction chip 100 and the sample detection chip 200 were placed in their respective positions.

[0076] The detection process is as follows: First, bacterial culture medium is added to nucleic acid extraction chip 100 using a pipette or syringe. The first temperature control module 300 heats the nucleic acid extraction chip 100 at 75 ℃ for 30 min for thermal lysis. Then, it is washed multiple times with purification solution, and the washing liquid is introduced into waste liquid collection device 706. Next, elution solution is added to elute the nucleic acid on glass fiber filter paper 107, and the elution liquid is introduced into sample detection chip 200 under the action of liquid guide tube. Finally, the second temperature control module 400 keeps the sample detection chip 200 at 37 ℃ for isothermal amplification and detection of nucleic acid. The fluorescence signal image is recorded in real time using the video function of a mobile phone, and the video is quantitatively analyzed using a self-developed program.

[0077] Finally, it should be noted that the above embodiments and examples are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments and examples, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments or examples, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments or examples of the present invention.

Claims

1. A nucleic acid extraction chip, characterized in that, The system comprises, from top to bottom, a flow divider layer, a filter layer, and a flow convergence layer that are tightly fitted together. The flow divider layer has an inlet, which is connected to multiple flow dividers via multiple first microchannels. The filter layer includes filter paper for enriching nucleic acids. The flow convergence layer has an outlet, which is connected to multiple flow convergence ports via multiple second microchannels. The number and distribution of the flow convergence ports correspond to the flow dividers. The inlet, the first microchannels, the flow dividers, the flow convergence ports, the second microchannels, and the outlet are sequentially connected, and the filter paper is disposed between the flow convergence ports and the flow dividers. The filter layer also includes two support layers, on which multiple support units are provided. The number and distribution of the support units correspond to the diversion port and the convergence port. Each support unit is provided with multiple support columns and through holes at intervals. The through holes are used to connect to the convergence port or the diversion port. The support units on the two support layers are arranged opposite to each other and clamp the filter paper. For the upper support layer, its support unit is perpendicular to the top diversion port in position and larger in area than the corresponding diversion port, and its through hole is connected to the diversion port; for the lower support layer, its support unit is perpendicular to the bottom convergence port in position and larger in area than the corresponding convergence port, and its through hole is connected to the convergence port.

2. The nucleic acid extraction chip according to claim 1, characterized in that, The filter paper is glass fiber filter paper or Whatman No.1 filter paper, with a pore size of 0.5~1.0μm.

3. A bacterial microfluidic integrated detection platform, characterized in that, It includes a bacterial lysis unit and a nucleic acid amplification reaction unit; the bacterial lysis unit includes a nucleic acid extraction chip as described in claim 1 or 2 and a first temperature control module, the first temperature control module being used to control the temperature of the nucleic acid extraction chip; the nucleic acid amplification reaction unit is used to perform isothermal amplification and detection of the nucleic acid extracted by the bacterial lysis unit.

4. The bacterial microfluidic integrated detection platform according to claim 3, characterized in that, The nucleic acid amplification reaction unit includes a sample detection chip and a second temperature control module. The second temperature control module is used to control the temperature of the sample detection chip, and the sample detection chip is used to amplify and detect the nucleic acid extracted by the bacterial lysis unit at an isothermal temperature.

5. The bacterial microfluidic integrated detection platform according to claim 4, characterized in that, The sample detection chip has an injection port, which is connected to at least one pair of parallel reaction detection ports and positive and negative control ports via a third microchannel. The reaction detection ports are pre-embedded with RPA nucleic acid amplification reagent, which includes RPA buffer, specific primers for the target bacterial gene, and fluorescent probes.

6. The bacterial microfluidic integrated detection platform according to claim 5, characterized in that, The RPA nucleic acid amplification reagent is freeze-dried at -80°C and then encapsulated in the reaction detection wells.

7. The bacterial microfluidic integrated detection platform according to claim 4, characterized in that, It also includes a housing and a signal reading unit; the signal reading unit includes a fluorescence excitation light source and an image acquisition device; the fluorescence excitation light source and the nucleic acid amplification reaction unit are disposed inside the housing, and the image acquisition device is used to acquire fluorescence signals during the isothermal amplification and inspection process of nucleic acid.

8. The bacterial microfluidic integrated detection platform according to claim 7, characterized in that, The angle between the light beam emitted by the fluorescent excitation source and the sample detection chip is 7~9°.

9. The bacterial microfluidic integrated detection platform according to claim 7 or 8, characterized in that, It also includes a first filter and a second filter. The first filter is disposed between the fluorescence excitation light source and the nucleic acid amplification reaction unit, and the transmission band of the first filter is the blue light band. The second filter is disposed between the nucleic acid amplification reaction unit and the image acquisition device, and the transmission band of the second filter is the green light band.

10. The bacterial microfluidic integrated detection platform according to claim 7, characterized in that, The housing includes a lower housing and an upper housing; the lower housing is used to house the bacterial lysis unit and the nucleic acid amplification reaction unit; the upper housing is fastened to the lower housing, the fluorescence excitation light source is disposed inside the upper housing and located above and to the side of the nucleic acid amplification reaction unit, and a through hole is also provided on the upper housing, the through hole being located directly above the nucleic acid amplification reaction unit, and the image acquisition device is disposed outside the housing and acquires the fluorescence intensity during the isothermal amplification and inspection process of nucleic acid through the through hole.

11. The bacterial microfluidic integrated detection platform according to claim 10, characterized in that, The lower housing is provided with a first loading module, a second loading module, a liquid guide pipe, and a waste liquid collection device; The first loading module includes a first mounting slot with an upper opening and a second mounting slot with a side opening; the first mounting slot is used to mount the nucleic acid extraction chip, and a drain port is provided at the bottom of the first mounting slot; the second mounting slot is used to mount the first temperature control module. The second loading module is disposed to the side and below the first loading module. The second loading module includes a third mounting slot with an upper opening and a fourth mounting slot with a side opening. The third mounting slot is used to install the sample detection chip. The fourth mounting slot is used to install the second temperature control module. One end of the liquid guide tube is connected to the drain port, and the other end is connected to the waste liquid collection device or the sample detection chip.

12. The bacterial microfluidic integrated detection platform according to claim 4, characterized in that, Both the first temperature control module and the second temperature control module include a silicone rubber heating plate and a heating controller connected to each other.

13. A method for detecting bacteria, characterized in that, The bacterial microfluidic integrated detection platform according to any one of claims 3-12 is used for detection. First, the bacterial culture medium is injected into the nucleic acid extraction chip, and after heating, lysis, purification and elution, nucleic acid is obtained. The obtained nucleic acid is introduced into the nucleic acid amplification reaction unit for isothermal amplification and detection.

14. The bacterial detection method according to claim 13, characterized in that, Decomposition was performed by isothermal heating at 70~100℃ for at least 30 min; isothermal amplification and detection were performed by isothermal heating at 37~42℃ for 10~20 min.

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