Microfluidic chip for diagnosing avian influenza h3 / h5 / h6 / h9 molecular typing based on microfluidics and use method thereof

Abstract

The application provides a microfluidic chip for diagnosing avian influenza H3 / H5 / H6 / H9 molecular typing based on microfluidics and a use method, relates to the field of pathogenic microorganism testing technology, and improves the efficiency, accuracy and convenience of avian influenza molecular diagnosis, promotes the development of molecular diagnosis technology, and uses the microfluidic chip body to be supported and used through the chip support, opens the protection layer attached to the microfluidic chip body by means of the light-proof assembly on the side, and can provide a light-proof environment again during and after the detection process, and can maintain a longer detection state.

Description

Technical Field

[0001] This invention relates to the field of pathogenic microorganism testing technology, specifically to a microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 and its usage method. Background Technology

[0002] The purpose of molecular typing of avian influenza H3 / H5 / H6 / H9 is to accurately identify viral subtypes and mutation characteristics, providing a basis for prevention and control. By distinguishing between highly pathogenic H3 / H5 and the potentially recombinant H9 subtype, precise culling, vaccination, and biosafety measures can be guided to block the transmission chain. Simultaneously, diagnostic testing can also analyze the genetic evolution of the virus, track key sites such as antigenic variation and drug resistance, and provide a data foundation for vaccine development and global epidemic monitoring.

[0003] Traditional molecular diagnostic methods currently used in technology have many shortcomings, such as cumbersome sample processing and detection steps, requiring multiple instruments and repeated manual operations, consuming a lot of time and manpower; the detection process is easily affected by environmental and human factors, resulting in poor accuracy and repeatability of results. While existing microfluidic chip-based detection can achieve a rapid and convenient typing and diagnostic process, it has disadvantages such as short post-test storage time and insufficient sealing after use. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 and its usage method, thereby solving the problems mentioned in the background. The present invention improves the efficiency, accuracy, and convenience of molecular diagnosis of avian influenza, promotes the development of molecular diagnostic technology, and the microfluidic chip body is supported and used by a chip scaffold. The protective layer attached to the microfluidic chip body can be opened by the light-shielding component on the side, so that a light-shielding environment can be provided again during and after the detection process, while also preventing internal reagents and samples from being contaminated, and maintaining a longer detection period.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: a microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9, comprising a microfluidic chip body and a matching chip support. The microfluidic chip body includes a substrate, a protective layer, and a sealing mechanism. The chip support includes a base plate, end plates, and a light-shielding component. The chip support has end plates integrally formed at both ends, with a retaining plate inserted at the top of one end plate. A positioning plate is mounted on the surface of the base plate, and support plates are attached to both sides of the positioning plate. The surface of the support plates is engraved with anti-slip textures. The microfluidic chip body is used to be embedded inside the chip support, and the positioning plate is used to provide support for the microfluidic chip body. A light-shielding component is mounted on the surface of the chip support, and the surface of the light-shielding component is used to press against the surface of the microfluidic chip body. A lead screw is inserted through the middle of the bottom of the light-shielding component, and the lead screw passes through the middle of one of the end plates.

[0006] Furthermore, a sealing layer is attached to the surface edge of the substrate, a protective layer is attached to the top of the sealing layer, a docking hole is provided at one end of the substrate, a microfluidic channel is provided on the inner side of the substrate, and a sample dispensing hole is provided at one end of the microfluidic channel.

[0007] Furthermore, a detection chamber is provided at the other end of the microfluidic channel, the protective layer is attached to the top of the sealing layer, the sealing mechanism includes an elastic sheet and a sealing sheet, the top of the sealing sheet is attached to the bottom of the elastic sheet, the sealing sheet is embedded in the interior of the docking hole, and the two ends of the elastic sheet are fixed to the sealing layer.

[0008] Furthermore, one of the end plates has a receiving groove on its top, the bottom of the card plate has an integrally formed plug plate, the top of the card plate has an overflow hole in the middle, the end plate has a through hole in the middle, and the lead screw passes through the inside of the through hole.

[0009] Furthermore, the card plate is embedded into the receiving groove through the bottom plug plate, and a vertical plate is fixed to the top of the other end plate. The top of the vertical plate is integrally formed with a top plate, and the middle of the top plate is integrally formed with an extension plate. A compression bolt is inserted in the middle of the extension plate, and a pressing pad is attached to the bottom end of the compression bolt. The pressing pad is used to press against the bottom protective layer, and the pressing pad is aligned with the sample feeding hole below.

[0010] Furthermore, the light-shielding component includes a sliding rod and an adhesive rod. The sliding rod has a threaded hole in the middle, and the lead screw passes through the inside of the threaded hole. Both ends of the sliding rod are screwed with linkage plates, and the top of the linkage plate is integrally formed with an arc-shaped top block. Both ends of the adhesive rod are equipped with rotating sleeves, and the arc-shaped top block is used to be embedded inside the rotating sleeve.

[0011] Furthermore, the outer side of the rotating sleeve is used to press against the surface of the tray, the bottom of the arc-shaped top block is fitted with a ball, and an annular groove is opened on the inner wall of the rotating sleeve. The arc-shaped top block is embedded into the groove through the ball at the bottom. The adhesive rod is bonded to one end of the protective layer through the adhesive layer on the surface.

[0012] A method of using the above-mentioned microfluidic chip for molecular typing diagnosis of avian influenza includes the following steps: S1. Pull the card plate portion at the end of the chip holder out of the end plate; S2. Push the microfluidic chip body horizontally to the inside of the chip holder and support it with the positioning plate; S3. Open the top protective layer using the light-blocking component; S4. Inject the sample into the syringe and connect the syringe to the sample dispensing port on the microfluidic chip body through the tube; S5. Inject the pretreated sample into the sample loading well, and the sample enters the detection chamber along the microfluidic channel; S6. Diagnose avian influenza H3 / H5 / H9 molecular typing by using four-color fluorescence color development.

[0013] Furthermore, in step S2, an adhesive is applied to the light-shielding component and bonded to one end of the protective layer on the chip. In step S4, the sample is a live poultry throat swab, and the blood sample is pre-mixed with an anticoagulant to prevent blockage of the microfluidic channels. The anticoagulant is sodium heparin. The sample is mixed with the chip-compatible lysis buffer and left to stand for 7 minutes to completely destroy the viral envelope while preserving the integrity of the RNA.

[0014] Furthermore, in step S5, a microfiltration membrane is built into the microfluidic channel to intercept blood cell impurities, allowing viral particles and free RNA to enter the chip core area. The captured viral RNA is reverse transcribed, and the reverse transcribed DNA flows into the detection chamber and comes into contact with three pre-implanted probes. These probes also serve as primers to specifically recognize target sequences of H3, H5, H6, and H9 subtypes. Each probe is connected to a fluorescent dye reagent of an independent color.

[0015] The beneficial effects of this invention are: 1. This invention effectively improves the efficiency, accuracy, and convenience of molecular diagnosis of avian influenza, promotes the development of molecular diagnostic technology, provides strong support for the rapid and accurate diagnosis of animal diseases, and has broad application prospects in the field of animal disease diagnosis.

[0016] 2. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 is supported and used by a chip scaffold. The protective layer attached to the microfluidic chip body is opened by the light-shielding component on the side. This allows the protective layer to cover and seal the substrate again after a period of time after the detection is completed, thus providing a light-shielding environment and reducing the detection sensitivity decay rate.

[0017] 3. This microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 applies pressure to the sample dispensing port at the front end through the top squeezing bolt and pressing pad, thereby providing a sealing effect during storage and after subsequent sample dispensing, preventing internal reagents and samples from being contaminated, and maintaining a longer detection period. Attached Figure Description

[0018] Figure 1 This is a diagram showing the state of the microfluidic chip after installation. Figure 2 This is a schematic diagram of the chip support portion of the present invention; Figure 3 This is a split view of the card plate portion of the present invention; Figure 4 This is a schematic diagram of the structure of the microfluidic chip body of the present invention; Figure 5 for Figure 4 Enlarged view of region A in the middle; Figure 6 This is a schematic diagram of the light-shielding component of the present invention; Figure 7 This is a split view of the end of the light-shielding component of the present invention; Figure 8 This is a flowchart illustrating the method of using the microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to the present invention; In the diagram: 1. Chip holder; 2. Microfluidic chip body; 3. Light-shielding component; 4. Sealing mechanism; 5. Base plate; 6. Knob; 7. End plate; 8. Positioning plate; 9. Support plate; 10. Anti-slip texture; 11. Vertical plate; 12. Top plate; 13. Extension plate; 14. Extrusion bolt; 15. Pressing pad; 16. Through hole; 17. Clamping plate; 18. Overflow hole; 19. Insertion plate; 20. Receiving groove; 21. Substrate; 22. Sealing layer; 23. Sample dispensing hole; 24. Microfluidic channel; 25. Detection chamber; 26. Protective layer; 27. Elastic sheet; 28. Docking hole; 29. ​​Sealing sheet; 30. Sliding rod; 31. Linkage plate; 32. Lead screw; 33. Adhesive rod; 34. Rotating sleeve; 35. Arc-shaped top block. Detailed Implementation

[0019] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0020] Please see Figures 1 to 8 This invention provides the following technical solution: a microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H9, comprising a microfluidic chip body 2 and a matching chip support 1. The microfluidic chip body 2 includes a substrate 21, a protective layer 26, and a sealing mechanism 4. The chip support 1 includes a base plate 5, end plates 7, and a light-shielding component 3. The two ends of the chip support 1 are integrally formed with end plates 7, and a retaining plate 17 is inserted into the top of one of the end plates 7. A positioning plate 8 is installed on the surface of the base plate 5, and support plates 9 are attached to both sides of the positioning plate 8. The surface of the support plates 9 is engraved with anti-slip textures 10. The microfluidic chip body 2 is used to be embedded into the inner side of the chip support 1, and the positioning plate 8 is used to provide support for the microfluidic chip body 2. The surface of the chip support 1 is equipped with a light-shielding component 3, and the surface of the light-shielding component 3 is used to press against the surface of the microfluidic chip body 2. A lead screw 32 is inserted through the middle of the bottom of the light-shielding component 3, and the lead screw 32 passes through the middle of one of the end plates 7. This microfluidic chip for typing diagnosis is used to perform molecular typing of avian influenza in samples.

[0021] In use, the card plate 17 at the end of the chip holder 1 is pulled out from the end plate 7, so that one end of the chip holder 1 is in an open state; the microfluidic chip body 2 is horizontally pushed to the inside of the chip holder 1 and supported by the positioning plate 8; the light-shielding component 3, initially located at one end, is pulled toward the end where the card plate 17 is located, and is bonded to one end of the protective layer 26 on the chip by applying adhesive to the light-shielding component 3; the top protective layer 26 is opened by the light-shielding component 3 until the protective layer 26 is rolled up to the bottom of the top plate 12, and the sample application port 23 is opened; the sample is injected into the syringe, and the syringe is connected to the sample application port 23 on the microfluidic chip body 2 through the tube. The sample is a live poultry throat swab, and the blood sample needs to be premixed with an anticoagulant to prevent the microfluidic channel from being blocked. Heparin sodium was used. The sample was mixed with the chip-compatible lysis buffer and allowed to stand for 7 minutes to completely destroy the viral envelope while preserving the RNA integrity. The pretreated sample was injected into sample well 23, and the sample entered the detection chamber 25 along the microfluidic channel 24. Inside the microfluidic channel 24, a built-in microfiltration membrane intercepted blood cell impurities, allowing viral particles and free RNA to enter the chip core area. The captured viral RNA underwent reverse transcription, and the reverse-transcribed DNA flowed into the detection chamber 25, where it came into contact with three pre-implanted probes. These probes also served as primers for specifically recognizing target sequences of H3, H5, and H9 subtypes. Each probe was linked to an independently colored fluorescent dye reagent. When DNA bound to the corresponding probe, the fluorescence signal intensity increased exponentially. The four-color fluorescence chromatographic state was used to diagnose avian influenza H3 / H5 / H6 / H9 molecular typing. The primer and probe sequences used for avian influenza (H3, H5, H6, H9 subtypes) and internal reference gene detection are shown in the table below. Table 1: Primer and probe sequence list for avian influenza (H3, H5, H6, H9 subtypes) and internal reference gene detection.

[0022] In this embodiment, a sealing layer 22 is attached to the surface edge of the substrate 21, and a protective layer 26 is attached to the top of the sealing layer 22. A docking hole 28 is provided at one end of the substrate 21, and a microfluidic channel 24 is provided on the inner side of the substrate 21. A sample application hole 23 is provided at one end of the microfluidic channel 24. A detection chamber 25 is provided at the other end of the microfluidic channel 24. The protective layer 26 is attached to the top of the sealing layer 22. The sealing mechanism 4 includes an elastic sheet 27 and a sealing sheet 29. The top of the sealing sheet 29 is attached to the bottom of the elastic sheet 27, and the sealing sheet 29 is embedded inside the docking hole 28. Both ends of the elastic sheet 27 are fixed to the sealing layer 22. Specifically, the injected sample is transported inside the substrate 21 through the microfluidic channel 24. The sample is injected from the sample loading hole 23 at one end and flows along the microfluidic channel 24 before entering the three sets of detection chambers 25 at the end. During the pushing process, the corresponding sample is discharged along the docking hole 28 and the overflow hole 18. After the detection is completed, the protective layer 26 is covered on the top of the microfluidic chip body 2 again by the light shielding component 3. It is then pressed on the top of the elastic sheet 27 and the sealing sheet 29 is pressed down to block the docking hole 28, so as to avoid external factors from interfering with the interior of the microfluidic channel 24.

[0023] In this embodiment, one end plate 7 has a receiving groove 20 on its top, and the bottom of the clamping plate 17 is integrally formed with a plug-in plate 19. The top center of the clamping plate 17 has an overflow hole 18, and the center of the end plate 7 has a through hole 16. The lead screw 32 passes through the inside of the through hole 16. The clamping plate 17 is embedded into the receiving groove 20 through the plug-in plate 19 at the bottom. The top of the other end plate 7 is fixed with a vertical plate 11. The top of the vertical plate 11 is integrally formed with a top plate 12. The center of the top plate 12 is integrally formed with an extension plate 13. A compression bolt 14 is inserted into the center of the extension plate 13. A pressing pad 15 is attached to the bottom end of the compression bolt 14. The pressing pad 15 is used to press against the bottom protective layer 26, and the pressing pad 15 is aligned with the sample feeding hole 23 below. Pressure is applied to the sample loading port 23 at the front end by the top compression bolt 14 and the pressing pad 15, which provides a sealing effect during storage and after subsequent sample loading, preventing internal reagents and samples from being contaminated and maintaining a longer detection period.

[0024] Specifically, during the long-term storage of the microfluidic chip body 2, the pressing pad 15 is pressed downward by rotating the compression bolt 14 until it rests against the protective layer 26. The protective layer 26 then presses against the sample application hole 23 directly below, thus sealing the sample application hole 23. After the protective layer 26 is unfolded by the light-shielding component 3, the external needle and micro-channel can be directly connected to the sample application hole 23. The end plate 7 of the card plate 17 is opened by plugging it in, providing a channel for the microfluidic chip body 2 to be inserted. It is also used to connect with the docking hole 28, and the discharged sample is discharged through the overflow hole 18.

[0025] In this embodiment, the light-shielding component 3 includes a sliding rod 30 and an adhesive rod 33. The sliding rod 30 has a threaded hole in the middle, through which a lead screw 32 passes. Both ends of the sliding rod 30 are screwed with linkage plates 31. An arc-shaped top block 35 is integrally formed on the top of the linkage plate 31. Rotating sleeves 34 are installed at both ends of the adhesive rod 33. The arc-shaped top block 35 is used to embed into the interior of the rotating sleeve 34. The outer side of the rotating sleeve 34 is used to press against the surface of the support plate 9. A ball bearing is embedded at the bottom of the arc-shaped top block 35, and an annular groove is formed on the inner wall of the rotating sleeve 34. The arc-shaped top block 35 is embedded into the groove through the ball bearing at its bottom. The adhesive rod 33 is bonded to one end of the protective layer 26 through an adhesive layer on its surface. Supported and used by the chip holder 1, the protective layer 26 attached to the microfluidic chip body 2 is opened with the help of the light-shielding component 3 on the side. This allows the protective layer 26 to cover and seal the substrate 21 again after a period of time after the detection is completed, thus providing a light-shielding environment again and reducing the detection sensitivity attenuation rate.

[0026] Specifically, after the microfluidic chip body 2 is installed, the protective layer 26 is first attached to the adhesive rod 33 with adhesive. Then, by turning the knob 6, the lead screw 32 is rotated. The threaded hole on the lead screw 32 and the sliding rod 30 are engaged, which controls the entire light-shielding assembly 3 to move along the surface of the base plate 5. During this process, the arc-shaped top block 35 at the end applies a pushing force inside the rotating sleeve 34, pushing the rotating sleeve 34 until it rolls along the support plate 9 towards the other end, thereby driving the adhesive rod 33 to rotate synchronously, realizing the winding process of the protective layer 26 until the bottom substrate 21 is completely opened, and the subsequent testing process can be carried out. After the testing is completed, the knob 6 is turned in the opposite direction to reset the light-shielding assembly 3 and the protective layer 26, and the entire substrate 21 is shielded from light again.

[0027] This embodiment also provides a method for using the above-mentioned microfluidic chip for avian influenza H3 / H5 / H9 molecular typing diagnosis, including the following steps: S1. Pull out the card plate 17 part at the end of the chip holder 1 from the end plate 7, so that one end of the chip holder 1 is in the open state. S2. Push the microfluidic chip body 2 horizontally to the inside of the chip holder 1 and support it with the positioning plate 8. Pull the light shielding component 3, which is initially located at one end, toward the end where the card plate 17 is located. Then, apply adhesive to the light shielding component 3 and bond it to one end of the protective layer 26 on the chip. S3. Open the top protective layer 26 through the light-shielding component 3 until the protective layer 26 is rolled up to the bottom of the top plate 12, and open the sample feeding hole 23. S4. Inject the sample into the syringe and connect the syringe to the sample application port 23 on the microfluidic chip body 2 through the tube. The sample is a live poultry throat swab. Blood samples need to be premixed with sodium heparin to prevent microfluidic channel blockage. The anticoagulant is sodium heparin. Mix the sample with the chip-compatible lysis buffer and let it stand for 7 minutes to completely destroy the viral envelope while preserving the integrity of the RNA. S5. Inject the pretreated sample into the sample well 23. The sample enters the detection chamber 25 along the microfluidic channel 24. Inside the microfluidic channel 24, the built-in micro-filter membrane intercepts blood cell impurities and allows virus particles and free RNA to enter the core area of ​​the chip. S6. The captured viral RNA is reverse transcribed, and the reverse transcribed DNA flows into the detection chamber 25, where it comes into contact with three pre-implanted probes. Each probe is connected to a fluorescent dye reagent of an independent color. When the DNA binds to the corresponding probe, the fluorescence signal intensity increases exponentially. The molecular typing of avian influenza H3 / H5 / H6 / H9 is diagnosed by the four-color fluorescence color development state.

[0028] This method effectively improves the efficiency, accuracy, and convenience of molecular diagnosis of avian influenza, promotes the development of molecular diagnostic technology, provides strong support for the rapid and accurate diagnosis of animal diseases, and has broad application prospects in the field of animal disease diagnosis.

[0029] The foregoing has shown and described the basic principles and main features of the present invention and its advantages. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention.

[0030] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9, comprising a microfluidic chip body (2) and a matching chip support (1), characterized in that: The microfluidic chip body (2) includes a substrate (21), a protective layer (26), and a sealing mechanism (4). The chip support (1) includes a base plate (5), an end plate (7), and a light-shielding component (3). The chip support (1) has an end plate (7) integrally formed at both ends. A card plate (17) is inserted into the top of one of the end plates (7). A positioning plate (8) is installed on the surface of the base plate (5). A support plate (9) is attached to both sides of the positioning plate (8). The surface of the support plate (9) is engraved with anti-slip embossing (10). The microfluidic chip body (2) is used to be embedded into the inner side of the chip support (1), and the positioning plate (8) is used to provide support for the microfluidic chip body (2). A light-shielding component (3) is installed on the surface of the chip support (1). The surface of the light-shielding component (3) is used to press against the surface of the microfluidic chip body (2), and a lead screw (32) is inserted through the middle of the bottom of the light-shielding component (3). The lead screw (32) passes through the middle of one of the end plates (7).

2. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 1, characterized in that: A sealing layer (22) is attached to the surface edge of the substrate (21), a protective layer (26) is attached to the top of the sealing layer (22), a docking hole (28) is provided at one end of the substrate (21), a microfluidic channel (24) is provided on the inner side of the substrate (21), and a sample feeding hole (23) is provided at one end of the microfluidic channel (24).

3. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 2, characterized in that: The other end of the microfluidic channel (24) is provided with a detection chamber (25). The protective layer (26) is attached to the top of the sealing layer (22). The sealing mechanism (4) includes an elastic sheet (27) and a sealing sheet (29). The top of the sealing sheet (29) is attached to the bottom of the elastic sheet (27). The sealing sheet (29) is embedded in the inside of the docking hole (28). The two ends of the elastic sheet (27) are fixed on the sealing layer (22).

4. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 2, characterized in that: One of the end plates (7) has a receiving groove (20) on its top, and the bottom of the card plate (17) is integrally formed with a plug plate (19). The top of the card plate (17) has an overflow hole (18) in the middle, and the end plate (7) has a through hole (16) in the middle. The lead screw (32) passes through the inside of the through hole (16).

5. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 4, characterized in that: The card plate (17) is embedded into the receiving groove (20) through the bottom plug plate (19). The top of the other end plate (7) is fixed with a vertical plate (11). The top of the vertical plate (11) is integrally formed with a top plate (12). The middle of the top plate (12) is integrally formed with an extension plate (13). A compression bolt (14) is inserted in the middle of the extension plate (13). A pressing pad (15) is attached to the bottom end of the compression bolt (14). The pressing pad (15) is used to press against the bottom protective layer (26), and the pressing pad (15) is aligned with the sample feeding hole (23) below.

6. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 4, characterized in that: The light-shielding component (3) includes a sliding rod (30) and an adhesive rod (33). The sliding rod (30) has a threaded hole in the middle, and the lead screw (32) passes through the inside of the threaded hole. The two ends of the sliding rod (30) are screwed with linkage plates (31). The top of the linkage plate (31) is integrally formed with an arc-shaped top block (35). The two ends of the adhesive rod (33) are equipped with rotating sleeves (34), and the arc-shaped top block (35) is used to be embedded into the inside of the rotating sleeve (34).

7. The microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 according to claim 6, characterized in that: The outer side of the rotating sleeve (34) is used to press against the surface of the tray (9). The bottom of the arc-shaped top block (35) is fitted with a ball, and an annular groove is opened on the inner wall of the rotating sleeve (34). The arc-shaped top block (35) is embedded into the groove through the ball at the bottom. The adhesive rod (33) is attached to one end of the protective layer (26) through the adhesive layer on the surface.

8. A method of using the microfluidic chip for molecular typing diagnosis of avian influenza H3 / H5 / H6 / H9 as described in claim 1, characterized in that, Includes the following steps: S1. Pull the card plate portion at the end of the chip holder out of the end plate; S2. Push the microfluidic chip body horizontally to the inside of the chip holder and support it with the positioning plate; S3. Open the top protective layer using the light-blocking component; S4. Inject the sample into the syringe and connect the syringe to the sample dispensing port on the microfluidic chip body through the tube; S5. Inject the pretreated sample into the sample loading well, and the sample enters the detection chamber along the microfluidic channel; S6. Diagnose avian influenza H3 / H5 / H6 / H9 molecular typing by using four-color fluorescence color development.

9. The method of use according to claim 8, characterized in that: In step S2, the light-shielding component is coated with an adhesive and bonded to one end of the protective layer on the chip. In step S4, the sample is a live poultry throat swab. The blood sample is pre-mixed with an anticoagulant to prevent blockage of the microfluidic channels. The anticoagulant is sodium heparin. The sample is mixed with the chip-compatible proteinase K lysis buffer and left to stand for 7 minutes to completely destroy the viral envelope while preserving the integrity of the RNA.

10. The method of use according to claim 8, characterized in that: In step S5, a microfiltration membrane is built into the microfluidic channel to intercept blood cell impurities, allowing viral particles and free RNA to enter the core area of ​​the chip. The captured viral RNA is reverse transcribed, and the reverse transcribed DNA flows into the detection chamber and comes into contact with four pre-coated probes. These probes also serve as primers to specifically recognize the target sequences of H3, H5, H6, and H9 subtypes. Each probe is connected to a fluorescent dye reagent of an independent color.

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