Microfluidic chip kit for rapid nucleic acid detection on basis of recombinase isothermal amplification

Abstract

Provided is a microfluidic chip kit for rapid nucleic acid detection on the basis of recombinase isothermal amplification. A microfluidic chip is provided with a sample introduction chamber, buffer chambers, extraction chambers, splitting chambers, amplification chambers and microfluidic flow channels for realizing sequential communication of the chambers. A corresponding nucleic acid release reagent dry powder is provided in advance in the extraction chambers, and a corresponding isothermal amplification reagent dry powder is provided in advance in each amplification chamber, so that any one of six common pathogens in swine herds can be amplified respectively. In addition, the pathogens amplified by the isothermal amplification reagent dry powders in all the amplification chambers can include the six pathogens, thereby achieving the detection requirement of simultaneously and rapidly detecting a plurality of pathogens.

Description

A microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202410066619.6 filed in China on January 17, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The invention belongs to the field of molecular biology, and in particular relates to a microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification. Background Art

[0004] In recent years, the rapid development of animal husbandry has led to increasing scale and commercialization. However, the occurrence of various animal diseases during the breeding process has always been a major hidden danger in animal husbandry production. Among them, animal infectious diseases are extremely harmful epidemic diseases caused by various pathogens. They can spread rapidly among livestock and are animal plagues with certain clinical symptoms and incubation periods. For example:

[0005] Porcine epidemic diarrhea (PED) is a serious, acute, contagious viral enteric disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV can persist in pig herds, affecting all ages. Therefore, once infected, pigs can suffer significant economic losses.

[0006] Highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) is an acute viral infectious disease caused by a variant strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive disorders in sows and respiratory diseases in piglets and fattening pigs. Pigs of different breeds, ages and genders are susceptible, with high morbidity and mortality rates, posing a serious threat to the pig industry.

[0007] Pseudorabies is an acute infectious disease of pigs caused by pseudorabies virus (PrV), and classical swine fever is an infectious disease caused by classical swine fever virus (CSFV). Pseudorabies and classical swine fever are the most common infectious diseases that cause reproductive disorders in pigs. Clinically, they sometimes present as mixed infections and have similar clinical manifestations, which makes clinical diagnosis very difficult.

[0008] Porcine circovirus disease is an infectious disease of pigs mainly caused by porcine circovirus (PCV) type 2, which seriously affects the development of the pig industry.

[0009] Swine flu is an acute respiratory disease caused by swine influenza virus (SIV). It not only seriously threatens the development of my country's pig industry, but also endangers human health.

[0010] Therefore, early detection of nucleic acids from these pathogens not only allows for understanding the infection status of pig farms, enabling early measures to control the outbreak, but also helps positive farms resume production. Currently, the most widely used method for detecting nucleic acids from these pathogens is the polymerase chain reaction (PCR). However, PCR requires expensive equipment, skilled technicians, and long testing times, making it difficult to scale up at the grassroots level or in resource-poor areas, and also prohibitive for on-site testing outside of laboratories. Developed isothermal nucleic acid amplification technologies simplify amplification conditions, eliminating the need for a thermal cycler and significantly reducing reliance on complex instrumentation. Recombinase polymerase amplification, a key component of isothermal nucleic acid amplification, is simpler to operate than other isothermal amplification technologies. It requires no initial heating or repeated enzyme additions during the reaction cycle, making it ideal for early, rapid clinical diagnosis and real-time on-site testing. On-site, real-time testing of clinical samples is also an effective means of preventing the rapid spread of disease. Rapid and reliable diagnostic tools also facilitate the implementation of appropriate treatments.

[0011] Nucleic acid isothermal amplification technology has low requirements for nucleic acid templates and does not require complete nucleic acid purification of the sample to be tested. Even if the sample source is complex, isothermal amplification technology still has good tolerance and can avoid conventional nucleic acid extraction steps. Microfluidic devices have the advantages of miniaturization and multi-channel sample detection. As a new technology platform, microfluidic chips integrate or substantially integrate basic operating units such as sample preparation, reaction, separation, and detection. Microchannels form a network, and controllable fluids run through the entire system. This reduces sample consumption, allows for rapid detection, and facilitates closed automated testing.

[0012] At present, the application of nucleic acid isothermal amplification in microfluidic chips mainly focuses on the rapid detection of nucleic acids using isothermal amplification technology with the help of microfluidic chips. It can be considered to combine with microfluidic chips to optimize the isothermal amplification technology to improve the efficiency, specificity and sensitivity of nucleic acid amplification reactions. At present, most of the research is mainly focused on the detection of a single pathogen, and for clinical cases suspected of mixed infection or similar clinical manifestations, rapid screening or disease typing cannot be achieved. In particular, the detection of multiple pathogens requires different detection kits for combined testing, which is time-consuming and costly, limiting the information that can be obtained from a single test. Based on isothermal amplification technology and microfluidic technology, a set of kits is provided that can quickly detect multiple pathogens at the same time, and can reduce the complexity of integrated nucleic acid extraction and amplification, meet the needs of nucleic acid-related testing and diagnosis, and have important practical significance for achieving simple and efficient nucleic acid diagnostic analysis.

[0013] It should also be noted that the above content falls within the technical knowledge of the inventor and does not necessarily constitute prior art. Summary of the Invention

[0014] The present invention provides a microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification, which meets the detection needs of simultaneous rapid detection of multiple pathogens, has good sensitivity and specificity, and nucleic acid extraction and amplification reactions are integrated on a single chip, which simplifies the operation process, saves time, and reduces detection costs.

[0015] The technical solution adopted by the present invention to solve the above technical problems is:

[0016] A microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification comprises a microfluidic chip, wherein the microfluidic chip is provided with an injection chamber, an amplification chamber, and a microfluidic flow channel for achieving communication between the injection chamber and the amplification chamber. The microfluidic chip comprises at least six amplification chambers, each of which is independent of each other and is pre-installed with a corresponding isothermal amplification reagent powder. The isothermal amplification reagent powder pre-installed in the amplification chamber can respectively amplify any one of the following six pathogens. At the same time, the pathogens amplified by the isothermal amplification reagent powder in all the amplification chambers can include the following six pathogens: porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus, classical swine fever virus, and swine influenza virus.

[0017] As an improved solution of the present invention, the isothermal amplification reagent dry powder includes primers, probes, DNA helicase and DNA polymerase for isothermal amplification of the six pathogens of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus, classical swine fever virus and swine influenza virus.

[0018] As an improved solution of the present invention, a buffer chamber, an extraction chamber and a shunt chamber are further arranged between the injection chamber and the amplification chamber of the microfluidic chip, and the microfluidic channel is arranged in sections to achieve sequential communication among the injection chamber, the buffer chamber, the extraction chamber, the shunt chamber and the amplification chamber.

[0019] As an improved solution of the present invention, the extraction chamber is pre-installed with corresponding nucleic acid releasing reagent dry powder.

[0020] As an improved solution of the present invention, the outermost layer of the microfluidic chip is a shell, and the inside of the shell is composed of a top plate, a fluid plate and a bottom plate from top to bottom.

[0021] As an improved solution of the present invention, the microfluidic flow channel includes a sample separation flow channel, a buffer flow channel, an extraction flow channel and an amplification flow channel arranged on the fluid plate. The injection chamber is connected to the buffer chamber through the sample separation flow channel, the buffer chamber is connected to the extraction chamber through the buffer flow channel, the extraction chamber is connected to the diversion chamber through the extraction flow channel, and the diversion chamber is connected to each amplification chamber through the amplification flow channel.

[0022] As an improved solution of the present invention, the injection chamber is arranged in the middle position of one side of the fluid plate, the buffer chamber is provided in two, and the two buffer chambers are respectively connected to the two sides of the injection chamber through the sample separation channel, the extraction chamber is provided in two, and the two extraction chambers are respectively connected to the corresponding buffer chambers through the buffer channel, the diversion chamber is provided in two, and the two diversion chambers are respectively connected to the corresponding extraction chambers through the extraction channel, and the top plate and the shell are respectively provided with injection ports corresponding to the injection chambers.

[0023] As an improved solution of the present invention, the amplification chambers are set into two groups, which are respectively located at the two ends of the fluid plate opposite to the injection chamber. The ends of each group of amplification chambers are arranged in sequence parallel to the fluid plate. The amplification flow channels are connected to the amplification chambers one by one, and each amplification chamber is connected to the corresponding diversion chamber through the amplification flow channels.

[0024] As a further improvement of the present invention, the extraction chamber includes an active mixing zone, a dry powder pre-setting zone and an overflow zone. A mixing step is provided in the active mixing zone. The base surface of the dry powder pre-setting zone is lower than the lowest mixing table surface of the active mixing zone. The overflow zone is arranged on one side of the active mixing zone and the dry powder pre-setting zone. The base surface of the overflow zone is higher than the highest mixing table surface of the active mixing zone. An overflow hole is provided in the overflow zone.

[0025] As a further improvement of the present invention, the extraction flow channel is a serpentine flow channel, the buffer flow channel is connected to the active mixing area, and the extraction flow channel is connected to the dry powder pre-placement area.

[0026] As a further improvement of the present invention, a waste liquid chamber and a clearance groove are provided in the bottom plate, an absorption pad is provided in the waste liquid chamber, and the overflow hole is communicated with the waste liquid chamber; the clearance groove is located below the amplification chamber.

[0027] As a further improvement of the present invention, the fluid plate is provided with ventilation holes for exhausting the waste liquid chamber, an air-permeable and water-blocking filter membrane is attached to the upper part of the ventilation holes, and ventilation openings are provided on the shell and the top plate corresponding to the ventilation holes.

[0028] As a further improvement of the present invention, the top plate is further provided with an air flow channel communicating with the diversion chamber and an air chamber communicating with the air flow channel, and a negative pressure inlet is provided on the shell corresponding to the air chamber.

[0029] As a further improvement of the present invention, the fluid plate is provided with a fluid infusion chamber and a bypass flow channel for replenishing liquid, the shell is provided with a fluid infusion tube, the end of the fluid infusion tube is communicated with the fluid infusion chamber on the fluid plate, the shell and the surface of the top plate have openings for the end of the fluid infusion tube to pass through, the end of the fluid infusion tube is sealed with a dissolving membrane, and a sealing cover is provided at the top of the fluid infusion tube. The upper section of the fluid infusion tube is a corrugated tube, and the lower section is conical, with the end facing the fluid infusion chamber of the fluid plate. A needle-shaped object is provided at the center of the end opposite to the fluid infusion chamber of the fluid plate, and the bypass flow channel connects the fluid infusion chamber and the two extraction flow channels.

[0030] As a further improvement of the present invention, a heat isolation area is further provided on the fluid plate, and the heat isolation area includes a plurality of heat isolation grooves, and the plurality of heat isolation grooves respectively surround the amplification flow channel and the amplification chamber.

[0031] As a further improvement of the present invention, a label covering area is provided on the surface of the shell, and a label is attached to the label covering area; a sample indicating area is provided on the shell, and the sample indicating area is located next to the sample injection chamber; and a detection port is provided on the shell.

[0032] As a further improvement of the present invention, a normally open stop valve is provided on the amplification flow channel, and the stop valve includes a valve seat, a valve core and a valve membrane. The valve seat is communicated with the amplification flow channel, and the valve core can produce a micro-displacement downward relative to the valve seat; the valve seat includes a diaphragm base and multiple valve seat body units connected together, the diaphragm base is fixed on the top plate, the number of valve seat body units is the same as the number of amplification chambers, the valve membrane is fixed between the valve seat body unit and the diaphragm base, and the valve membrane corresponds to the valve seat body unit one by one. When the stop valve is closed, the valve core and the valve membrane cut off the amplification flow channel.

[0033] As a further improvement of the present invention, a normally closed shut-off valve is provided on the extraction flow channel, and the shut-off valve includes a shut-off valve seat, a shut-off valve core and a shut-off valve membrane. The shut-off valve seat is communicated with the extraction flow channel, and the shut-off valve core can produce a micro-displacement downward relative to the shut-off valve seat; the shut-off base is fixed on the top plate, and the shut-off valve membrane and the lower end of the shut-off valve core are wrapped as one body. When the shut-off valve is opened, the extraction flow channel is communicated with the diversion chamber.

[0034] As a further improvement of the present invention, the sequences of the primers are: porcine epidemic diarrhea virus, SEQ ID NO.1-2; porcine reproductive and respiratory syndrome virus, SEQ ID NO.4-5; pseudorabies virus, SEQ ID NO.7-8; porcine circovirus, SEQ ID NO.10-11; classical swine fever virus, SEQ ID NO.13-14; swine influenza virus, SEQ ID NO.16-17; the sequences of the probes are: porcine epidemic diarrhea virus, SEQ ID NO.3; porcine reproductive and respiratory syndrome virus, SEQ ID NO.6; pseudorabies virus, SEQ ID NO.9; porcine circovirus, SEQ ID NO.12; classical swine fever virus, SEQ ID NO.15; swine influenza virus, SEQ ID NO.18.

[0035] As a further improvement of the present invention, two of the amplification chambers on the microfluidic chip are set as a positive control chamber and a negative control chamber, wherein the positive control chamber is pre-installed with a positive quality control product, and the positive quality control product contains plasmids corresponding to the amplified gene sequences of the above six pathogens.

[0036] Beneficial effects of the present invention:

[0037] The microfluidic chip kit provided by the present invention can enable liquid samples to flow through each chamber in an orderly manner, independently of each other, and without interfering with each other, through the sampling chamber, buffer chamber, extraction chamber, shunt chamber and amplification chamber, as well as the microfluidic flow channel that connects the sampling chamber, buffer chamber, extraction chamber, shunt chamber and amplification chamber in sequence.

[0038] The kit of the present invention combines constant-temperature amplification technology with a microfluidic chip to simultaneously detect six pathogens that pigs are susceptible to; it has strong anti-interference ability, and good amplification results can be obtained with a crude DNA template; using the matching nucleic acid release reagent dry powder, DNA extraction can be completed quickly (5 minutes) in a non-experimental environment, and using the matching constant-temperature amplification reagent dry powder, amplification can be completed quickly (20 minutes) at near body temperature (39°C), reducing the number of operating steps, saving time, and having low technical requirements for operators; avoiding sample contamination that may be caused during the experiment, and the test results are stable and reliable, with good reproducibility, strong specificity, and high sensitivity.

[0039] The microfluidic chip of the present invention integrates nucleic acid extraction and DNA amplification. The detection process is a closed reaction, which reduces the number of experimental steps, reduces the risk of contamination and the possibility of deviation in results. In addition, the entire system does not contain harmful substances, and the amplified products do not require special post-processing.

[0040] The extraction chamber structure of the microfluidic chip of the present invention ensures efficient reaction mixing, and the rehydration chamber can promptly replenish reaction reagents, fully redissolve dry powder reagents, ensuring sufficient mixed liquid for stable amplification during amplification. The waste liquid chamber prevents fluid leakage during detection, posing no risk to operators or the environment. The addition of a stop valve eliminates the risk of leakage even if the chip is inverted or malfunctions, enhancing safety while preventing crosstalk between amplification chambers. The addition of a shutoff valve maintains a negative pressure state in the chip in advance, eliminating the need for external drivers, reducing device complexity, and facilitating real-time detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention.

[0042] FIG2 is a schematic structural diagram of a housing according to an embodiment of the present invention.

[0043] FIG3 is a schematic diagram of a microfluidic chip without a housing structure according to an embodiment of the present invention.

[0044] FIG4 is a schematic diagram of the top plate structure of an embodiment of the present invention.

[0045] FIG5 is a schematic diagram of the structure of a fluid plate according to an embodiment of the present invention.

[0046] FIG. 6 is a front view of a fluid plate according to an embodiment of the present invention.

[0047] FIG7 is a schematic structural diagram of a bottom plate according to an embodiment of the present invention.

[0048] FIG8 is a simplified diagram illustrating the functions of the microfluidic chip according to an embodiment of the present invention.

[0049] FIG9 is a graph showing the specificity test results of the recombinase isothermal amplification method based on a microfluidic chip.

[0050] FIG10 is a diagram showing the sensitivity test results of the recombinase isothermal amplification method based on a microfluidic chip.

[0051] FIG11 is a diagram showing the repeatability test results of the recombinase isothermal amplification method based on a microfluidic chip.

[0052] FIG12 is a schematic diagram of a microfluidic chip without a housing structure according to another embodiment of the present invention.

[0053] FIG13 is a schematic diagram of the top plate structure of another embodiment of the present invention.

[0054] FIG14 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.

[0055] FIG15 is a cross-sectional view of a first stop valve structure in another embodiment of the present invention, wherein FIG15a shows the stop valve in an unclosed state, and FIG15b shows the stop valve in a closed state.

[0056] FIG16 is a schematic structural diagram of a first stop valve in another embodiment of the present invention.

[0057] FIG17 is a cross-sectional view of a second stop valve structure in another embodiment of the present invention, wherein FIG17a shows the stop valve in an unclosed state, and FIG17b shows the stop valve in a closed state.

[0058] FIG18 is a schematic diagram of a microfluidic chip without a housing structure according to another embodiment of the present invention.

[0059] FIG19 is a cross-sectional view of the shut-off valve structure in yet another embodiment of the present invention.

[0060] FIG20 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.

[0061] In Figure 1, 100. housing, 101. detection port, 102. housing vent, 103. sealing rubber plug port, 104. tube cover, 105. cover, 106. label covering area, 107. sample indication area, 200. top plate, 300. fluid plate, 400. bottom plate;

[0062] In Figure 2, 100. housing, 101. detection port, 102. housing vent, 103. sealing rubber plug port, 104. tube cover, 105. cap, 106. label covering area, 107. sample indication area, 108. housing negative pressure inlet, 109. wing, 110. housing sample inlet, 111. housing refill port, 112. refill tube;

[0063] In Figure 3, 200. top plate, 300. fluid plate, 400. bottom plate;

[0064] In Figure 4, 201. Top plate sample inlet, 202. Top plate vent, 203. Top plate refill port, 204. Air flow channel, 205. Gas chamber, 206. Top plate assembly hole;

[0065] In Figures 5 and 6, 300. fluid plate, 301. injection chamber, 302. sample distribution channel, 303. buffer chamber, 304. buffer channel, 305. extraction chamber, 3051. active mixing area, 3052. dry powder pre-setting area, 3053. overflow area, 3054. overflow hole, 306. extraction channel, 307. diversion chamber, 308. amplification channel, 309. amplification chamber, 310. ventilation hole, 311. rehydration chamber, 312. bypass channel, 313. thermal isolation groove, 314. needle, 315. fluid plate assembly hole;

[0066] In Figure 7, 400. Bottom plate, 401. Waste liquid chamber, 402. Groove, 403. Flat support, 404. Column, 405. Give way slot;

[0067] In Figure 8, 204. air flow channel, 205. air chamber, 301. sample injection chamber, 302. sample splitting channel, 303. buffer chamber, 304. buffer channel, 305. extraction chamber, 306. extraction channel, 307. split chamber, 308. amplification channel, 309. amplification chamber, 311. rehydration chamber, 312. bypass channel, 313. thermal isolation tank, 401. waste liquid chamber;

[0068] In Figure 12, 200. top plate, 300. fluid plate, 400. bottom plate, 500. stop valve;

[0069] In Figure 13, 201. Top plate sample inlet, 202. Top plate vent, 203. Top plate liquid filling port, 204. Air flow channel, 205. Gas chamber, 206. Top plate assembly hole, 207. Shut-off valve connection hole;

[0070] In Figure 14, 100. Housing, 101. Inspection port, 102. Housing vent, 103. Sealing rubber plug port, 104. Tube cover, 105. Cover, 106. Label covering area, 500. Stop valve, 200. Top plate, 300. Fluid plate, 400. Bottom plate;

[0071] In Figures 15a and 15b, 501. valve seat, 5011. valve seat body unit, 5012. diaphragm base, 502. valve core, 503. valve membrane, 504. valve cap, 200. top plate, 207. stop valve connection hole, 300. fluid plate, 308. amplification flow channel, 316. connection pool;

[0072] In Figure 16, 5011. Valve seat body unit, 5012. Base, 504. Valve cap, 505. Safety buckle;

[0073] In Figures 17a and 17b, 501. valve seat, 5011. valve seat body unit, 5012. diaphragm base, 502. valve core, 503. valve diaphragm, 504. valve cap, 506. flap, 507. ridge, 200. top plate, 207. stop valve connection hole, 300. fluid plate, 308. amplification flow channel, 316. connection pool;

[0074] In Figure 18, 200. Top plate, 300. Fluid plate, 400. Bottom plate, 600. Shutoff valve;

[0075] In Figure 19, 601. shut-off valve seat, 602. shut-off valve core, 603. shut-off valve membrane, 604. shut-off valve cap, 200. top plate, 2087. shut-off valve communication hole, 300. fluid plate, 308. amplification flow channel, 317. shut-off reservoir;

[0076] In Figure 20, 100. Shell, 101. Inspection port, 102. Shell vent, 103. Sealing rubber plug port, 104. Tube cover, 105. Cover, 106. Label covering area, 600. Shut-off valve, 200. Top plate, 300. Fluid plate, 400. Bottom plate. DETAILED DESCRIPTION

[0077] This invention combines constant-temperature amplification technology with microfluidic chip technology. A brief description of the constant-temperature amplification technology is now provided. Body Temperature Nucleic Acid Amplification (BTNAA) involves a recombinase and primer forming a protein / single-stranded nucleotide complex, Rec / ssDNA, at constant temperature. With the help of accessory proteins and the single-stranded binding protein SSB, the complex invades a double-stranded DNA template. A D-loop region is formed at the invasion site, and scanning of the DNA double strand begins. Upon finding the target region complementary to the primer, the Rec / ssDNA complex disintegrates, while the polymerase binds to the 3' end of the primer, initiating chain extension. This recombinase-polymerase constant-temperature amplification technology offers unique advantages as a rapid, convenient, and specific diagnostic method.

[0078] In order to better describe this embodiment, the following explanation is made: the upstream or downstream of the flow channel mentioned below refers to the section of the flow channel through which the liquid flows first as the upstream, and the section through which the liquid flows later as the downstream. Since the microfluidic chip requires a small amount of sample and has high requirements for signal detection, the supporting dedicated constant temperature fluorescence detection equipment mentioned below has the fluorescence detection function of the existing fluorescence detector, and the micro pump / syringe can be used as a pressure providing device. The terms mentioned below such as "inside", "outside", "left", "right", "up", "down", "side", "bottom", "front", "back" and the like indicate the orientation or position relationship based on the orientation or position relationship shown in the accompanying drawings, and are only relative terms determined for the convenience of describing the structural relationship of the various components or elements in the present invention, and should not be understood as limiting the present invention. The parts not described in detail in the present invention are all well-known technologies for those skilled in the art.

[0079] Example 1

[0080] As shown in Figures 1, 3 and 5, the microfluidic chip kit for rapid detection of nucleic acids based on recombinase isothermal amplification of the present invention includes a microfluidic chip, on which an injection chamber 301, a buffer chamber 303, an extraction chamber 305, a shunt chamber 307 and an amplification chamber 309 are provided, as well as a microfluidic flow channel that connects the injection chamber 301, the buffer chamber 303, the extraction chamber 305, the shunt chamber 307 and the amplification chamber 309 in sequence. The extraction chamber 305 is pre-installed with a corresponding nucleic acid release reagent powder, the main components of which are lithium dodecyl sulfate and NaCl. The six amplification chambers 309 are all pre-installed with a corresponding constant temperature amplification reagent powder. The constant temperature amplification reagent powder pre-installed in the amplification chambers 309 can respectively amplify any one of the following six pathogens. At the same time, the pathogens amplified by the constant temperature amplification reagent powder in all amplification chambers 309 can include the following six pathogens: porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PrV), porcine circovirus (PCV), classical swine fever virus (CSFV), and swine influenza virus (SIV). The constant temperature amplification reagent powder in the present invention includes primers, probes, DNA helicase, and DNA polymerase for constant temperature amplification of the above six pathogens.

[0081] The sequences of the primers are: porcine epidemic diarrhea virus, SEQ ID NO.1-2; porcine reproductive and respiratory syndrome virus, SEQ ID NO.4-5; pseudorabies virus, SEQ ID NO.7-8; porcine circovirus, SEQ ID NO.10-11; classical swine fever virus, SEQ ID NO.13-14; and swine influenza virus, SEQ ID NO.16-17.

[0082] The sequences of the probes are: porcine epidemic diarrhea virus, SEQ ID NO.3; porcine reproductive and respiratory syndrome virus, SEQ ID NO.6; pseudorabies virus, SEQ ID NO.9; porcine circovirus, SEQ ID NO.12; classical swine fever virus, SEQ ID NO.15; and swine influenza virus, SEQ ID NO.18.

[0083] The microfluidic chip has two amplification chambers 309 configured as a positive control chamber and a negative control chamber. These two amplification chambers 309 are located on different sides of the chip, such as on the left and right sides. The positive control chamber is pre-installed with a positive quality control product containing a plasmid corresponding to the amplified gene sequences of the six pathogens mentioned above. The negative control chamber is a blank chamber. As shown in Figure 3, due to the addition of the positive and negative control chambers, a total of eight amplification chambers 309 are arranged on the microfluidic chip, and each amplification chamber 309 is independent of each other.

[0084] The microfluidic chip according to the embodiment of the present invention will be described in detail below.

[0085] As shown in Figures 1 to 3, the top layer of the microfluidic chip in the embodiment of the present invention is a housing 100, and below the housing 100 are a top plate 200 and a fluid plate 300. Below the fluid plate 300 is a bottom plate 400, wherein: the upper portion of the fluid plate 300 is adhered to the bottom of the top plate 200, and the lower portion of the fluid plate 300 is adhered to the upper portion of the bottom plate 400. The bottoms of the two wings 109 of the housing 100 are embedded in the grooves 402 on the bottom plate 400. The two inner sides of the housing 100 can support the top plate 200 and the fluid plate 300, and the lower portion of the housing 100 can support the bottom plate 400. The microfluidic chip of the present invention can be used in conjunction with a constant temperature fluorescence detection device. The housing 100 is also provided with a detection port 101. The optical module of the constant temperature fluorescence detection device can emit excitation light from the detection port 101 into the product to be tested, thereby stimulating the product to react and produce fluorescence. The housing 100, top plate 200, and fluid plate 300 are all provided with assembly holes, such as the top plate assembly hole 206 on the top plate 200 and the fluid plate assembly hole 315 on the fluid plate 300. The bottom plate 400 is provided with a post 404 for limiting the assembly holes. The bottom plate 400 is also provided with a flat support 403 that contacts the top plate 200 and can also limit the position of the fluid plate 300. A label covering area 106 is provided on the right side of the surface of the housing 100, and a label is attached to the label covering area 106. The label can be a machine-readable QR code or barcode. After scanning, the computer can automatically read and identify it to obtain and / or determine the test number, test name, batch number and shelf life of the chip. To facilitate fluorescence detection and liquid display in the sample indicator area 107 on the fluid plate 300, the top plate 200, fluid plate 300, and bottom plate 400 in this embodiment can be made of transparent materials. The fluid plate 300 is mainly made of glass, quartz and plastic, and has good light transmittance. The housing 100 can not only protect the important main body of the chip, but also facilitate stable holding in the detection equipment.

[0086] As shown in Figures 5 and 6, the injection chamber 301 is located in the middle of the front side of the fluid plate 300. The buffer chambers 303 on the fluid plate 300 are respectively located on the left and right sides of the injection chamber 301, and the two buffer chambers 303 are symmetrically arranged relative to the injection chamber 301. This configuration of the injection chamber 301 and the two buffer chambers 303 can properly distribute the sample source when the sample amount is appropriate, and can also ensure that the subsequent reaction chambers are filled with sufficient amount when the sample amount is excessive. Even if the sample is added at a high speed, the two buffer chambers 303 can drain and store the sample reagents in the injection chamber 301 first, and the sample reagents can also be evenly distributed to the two buffer chambers 303. The extraction chamber 305 on the fluid plate 300 includes an active mixing area 3051, a dry powder pre-setting area 3052 and an overflow area 3053. The active mixing area 3051 is provided with multiple mixing steps. The base surface of the dry powder pre-setting area 3052 is lower than the lowest mixing table surface of the active mixing area 3051. The overflow area 3053 is arranged on one side of the active mixing area 3051 and the dry powder pre-setting area 3052. The base surface of the overflow area 3053 is higher than the highest mixing table surface of the active mixing area 3051. The lowest level of the multiple mixing steps is slightly inclined toward the overflow area 3053, and the two sides of each level are tangent to the side walls of the active mixing area 3051. The dry powder pre-positioning area 3052 is located at the downstream convex corner of the extraction chamber 305, which protrudes toward the rear of the chip. The two sides of the dry powder pre-positioning area 3052 are tangentially connected to the active flow mixing area 3051. The nucleic acid release reagent dry powder is fixed in the dry powder pre-positioning area 3052. The fixing method can be to heat and melt paraffin and spray it onto the surface of the reagent dry powder. After cooling and solidification, the reagent dry powder is fixed in the dry powder pre-positioning area 3052. In this embodiment, the active flow mixing area 3051 preferably has a cylindrical groove structure. The step profile curve of each mixing step in the active flow mixing area 3051 is a fitted circular arc curve. The overflow area 3053 has an overflow hole 3054. The buffer flow channel 304 is connected to the active flow mixing area 3051, and the extraction flow channel 306 is connected to the dry powder pre-positioning area 3052. When the sample reagent enters the extraction chamber 305, it first passes through the active flow mixing area 3051 for mixing before flowing into the dry powder pre-positioning area 3052. Active mixing zone 3051 disrupts stratified flow by introducing disturbances, enhancing intermixing between flow layers and increasing the probability of fluid contact, thereby achieving optimal mixing with the reagent powder. After securing the reagent powder, consider spraying paraffin wax in the fluid confluence area of ​​dry powder pre-position zone 3052 and allowing it to solidify to form a hydrophobic wall. This barrier acts as a barrier, increasing the reaction time of the sample reagent in dry powder pre-position zone 3052. However, the hydrophobic wall cannot completely prevent the reagent from flowing from the outlet of dry powder pre-position zone 3052 to the extraction channel 306.

[0087] After nucleic acid extraction from the sample, before the amplification reaction begins, the mixed liquid needs to be evenly distributed into the amplification channels 308 leading to the amplification chambers 309. To ensure that all amplification channels 308 are filled with liquid, the mixed liquid needs to be diverted through the diversion chambers 307. The diversion chambers 307 on the fluid plate 300 are long and narrow strips. The amplification chambers 309 on the fluid plate 300 are arranged in two groups, with each amplification chamber 309 in each group connected to a diversion chamber 307. The two groups of amplification chambers 309 are located on the left and right ends of the fluid plate 300, respectively, and the amplification chambers 309 of each group are arranged in sequence parallel to the left and right ends of the fluid plate 300.

[0088] As shown in Figures 5 and 6, the microfluidic channel includes a sample splitting channel 302, a buffer channel 304, an extraction channel 306, and an amplification channel 308. The two buffer chambers 303 are connected to the sample injection chamber 301 via the sample splitting channel 302, the two extraction chambers 305 are connected to the corresponding buffer chambers 303 via the buffer channel 304, the two diversion chambers 307 are connected to the corresponding extraction chambers 305 via the extraction channel 306, the upstream of the amplification channel 308 is connected to the diversion chamber 307, and the downstream of the amplification channel 308 is connected to the amplification chamber 309 in a one-to-one correspondence. The microfluidic channel is arranged in this segmented manner to achieve sequential communication between the sample injection chamber 301, the buffer chamber 303, the extraction chamber 305, the diversion chamber 307, and the amplification chamber 309.

[0089] As shown in Figures 2 and 4, the top plate 200 is provided with a top plate inlet 201 corresponding to the inlet chamber 301, and the housing 100 is provided with a housing inlet 110 corresponding to the inlet chamber 301. The extraction channel 306 is a serpentine channel. In this embodiment, the extraction channel 306 is a combination of a semicircular channel and a linear channel. By changing the geometry of the channel, mixing is achieved in a short period of time. When the mixed liquid flows through the semicircular channel, the flow direction changes, the liquids are further integrated, and the mixing in the span direction is enhanced.

[0090] As shown in Figure 7 , in this embodiment, each group of amplification chambers 309 consists of four. Directly below each group of amplification chambers 309 is a recess 405 in the base plate 400. Each group of four amplification chambers 309 is arranged sequentially along the edge of the chip to ensure that each amplification chamber 309 is independent and separated from each other to prevent cross-contamination. For reference, the recess 405 can provide a heat transfer area for the heating module of the accompanying constant-temperature fluorescence detection equipment to heat the amplification chambers 309; it can also provide an optical path collection area for the fluorescence detection module.

[0091] As shown in FIG1 , the sample indication area 107 on the housing 100 is positioned adjacent to the injection chamber 301. As shown in FIG3 and FIG4 , the top plate 200 is further provided with an air flow channel 204 communicating with the diversion chamber 307. An air chamber 205 is provided on the top plate 200 corresponding to the air flow channel 204, and a housing negative pressure inlet 108 is provided on the housing 100 corresponding to the air chamber 205. The housing negative pressure inlet 108 on the housing 100 is adapted to accommodate a sealing rubber plug 103. The housing injection port 110 is adapted to accommodate the tube cap 104.

[0092] As shown in Figure 7 , the outer edge of the top of the bottom plate 400 is adhered to the bottom of the fluid plate 300. The space between the fluid plate 300 and the bottom plate 400 forms a waste liquid chamber 401, which contains an absorbent pad. The overflow hole 3054 of the extraction chamber 305 on the fluid plate 300 communicates with the waste liquid chamber 401. The waste liquid chamber 401 is inclined toward the center from the periphery. As shown in Figure 5 , the fluid plate 300 has a vent hole 310 for exhausting the waste liquid chamber 401. A breathable and water-blocking filter membrane is attached to the top of the vent hole 310. As shown in Figure 2 , the housing 100 has a housing vent 102 corresponding to the vent hole 310, and the top plate 200 has a top plate vent 202 corresponding to the vent hole 310. A sealing film is attached to the top plate vent 202 to prevent bacteria while ensuring normal gas exchange.

[0093] As shown in Figures 2 and 3, the housing 100 is provided with a refill tube 112, and the fluid plate 300 is also provided with a refill chamber 311. The distal end of the refill tube 112 communicates with the refill chamber 311 on the fluid plate 300. Ports for the refill tube 112 to pass through are provided on the surfaces of the housing 100 and the top plate 200, such as the housing refill port 111 and the top plate refill port 203 (see Figure 4). The distal end of the refill tube 112 is sealed with a dissolving membrane, and a cap 105 is provided at the top of the refill tube 112. The upper section of the refill tube 112 is a corrugated tube, while the lower section is tapered. The distal end of the refill tube 112 faces the refill chamber 311 of the fluid plate 300. A needle 314 is provided within the refill chamber 311 of the fluid plate 300, directly opposite the distal end at the center. The liquid infusion chamber 311 is communicated with the bypass channel 312 , and the bypass channel 312 is configured in a Y-shape to be communicated with the downstream sections of the two extraction channels 306 respectively.

[0094] As shown in Figure 6, a thermal isolation area is also provided on the fluid plate 300, and the thermal isolation area includes a plurality of small thermal isolation grooves 313. These thermal isolation grooves surround the amplification flow channel 308 and the amplification chamber 309 respectively. A closed space is formed between the thermal isolation grooves 313 and the top plate 200, which is used to isolate the amplification flow channel 308 and the amplification chamber 309 from the surrounding area. The still air trapped in the closed space is a poor conductor of heat and has good thermal insulation properties, making it easy to maintain a constant temperature in the amplification chamber 309. When the heating module of the matching constant temperature fluorescence detection equipment can provide heating to each group of amplification chambers 309 from the give way groove 405, it will be more conducive to rapid amplification reaction.

[0095] The final concentration of the primers in the microfluidic chip kit of the present invention in the amplification system is 100-1000 nM; the final concentration of the probe in the amplification system is 50-500 nM.

[0096] The primer and probe sequence information is shown in Table 1 below. The modified probe sequence information is shown in Table 2 below.

[0097] Table 1 Primer and probe sequence information

[0098] .

[0099] Table 2 Primer and probe sequence information

[0100] .

[0101] Note: F is the upstream primer, R is the downstream primer, and P is the probe; FAM-dT refers to dT nucleotides fluorescently labeled with FAM (carboxyfluorescein); dSpacer refers to base deletion; BHQ1-dT refers to dT nucleotides labeled with a BHQ1 quencher; THF residues (tetrahydrofuran) are located between [FAM-dT] and [BHQ1-dT]; C3Spacer as a blocking group refers to 3′ hydroxyl blocking; P refers to phosphorylation.

[0102] Kit operation and result determination:

[0103] 1. Sample Collection and Processing

[0104] Sample collection is performed as needed.

[0105] (1) Oral swab sample processing: Oscillate the oral swab tube at 6000 r / min for 45 seconds and take 200 μL for later use.

[0106] (2) Processing of spleen, lung, kidney and other tissue samples: Take 0.05 g of tissue blocks and place them in a grinding tube filled with PBS buffer. Grind at 6000 r / min for 45 seconds to make a tissue homogenate of about 10%. Centrifuge at 5000 g for 5 minutes and take 200 μL of supernatant for later use.

[0107] (3) Fecal and feed sample processing: Take 1g of feces or feed and place it in a grinding tube filled with PBS buffer. Grind at 6000 r / min for 45 seconds to make a 10% homogenate. Centrifuge at 5000 g for 5 minutes and collect 200 μL of the supernatant for later use. (PBS: phosphate buffer saline)

[0108] (4) Sewage sample treatment: Take 200 μL of sewage for later use.

[0109] (5) Non-hemolyzed serum or plasma: no pretreatment required.

[0110] 2. Amplification reaction and detection:

[0111] (1) Open the tube cover of the shell sample inlet 110 on the microfluidic chip shell 100, and the sample flows into the sample injection chamber 301 from the shell sample inlet 110 and the top plate sample inlet 201. The sample in the sample injection chamber 301 flows into the sample splitting flow channels 302 on both sides and reaches the corresponding buffer chamber 303. The sample flow can be seen from the sample indicator area 107 on the shell 100, and then flows through the sample splitting flow channels 302 until all the samples flow into the buffer chamber 303. The existence of two buffer chambers 303 can not only distribute the sample evenly, but also increase the sample loading speed. After the sample loading is completed, close the tube cover 104 of the shell sample inlet 110. Allow the sample to be excessive so that the sample volume added to the sample injection chamber 301 is large enough to ensure proper filling during the amplification stage.

[0112] The ventilation holes 310 on the fluid plate 300 for exhausting the waste liquid chamber 401, the top plate vents 202 on the top plate 200, and the shell vents 102 on the shell 100 maintain the pressure balance between the waste liquid chamber 401 and the outside world. The waste liquid chamber 401 also vents the pressure in the fluid plate 300 to atmospheric pressure through the overflow hole 3054 of the extraction chamber 305 to ensure the balance of air pressure in the flow channel and avoid the negative impact of air pressure hindering the normal flow of samples in the fluid plate 300.

[0113] (2) The sample flows out of the buffer chamber 303 and enters the buffer flow channel 304, and then enters the extraction chamber 305. The sample passes through the mixing steps of the active mixing area 3051 and reaches the dry powder pre-setting area 3052 layer by layer, which increases the time the sample reagent stays in the dry powder pre-setting area 3052, and the nucleic acid release reagent dry powder can be fully redissolved to complete the nucleic acid extraction. The negative pressure inlet 108 of the chip shell is connected to the syringe to manually realize negative pressure drive. No external power source is required, the structural complexity is not increased, the device is easy to be miniaturized, and it can be well adapted to on-site timely detection. The gas in the fluid plate 300 flows out through the air flow channel 204 and the air chamber 205, the pressure in the diversion chamber 307 and the amplification flow channel 308 is reduced, and the liquid is driven into the serpentine part of the extraction flow channel 306 and mixed, thereby increasing the controllability of the reagent reaction and obtaining a nucleic acid template for pathogen detection. When the liquid in the extraction chamber 305 overflows excessively, it will enter the overflow groove 3053. Then, the excess liquid will flow into the waste liquid chamber 401 from the overflow hole 3054 in the overflow groove 3053 and be completely absorbed by the absorption pad.

[0114] (3) The sample (nucleic acid template) flows evenly into each diversion chamber 307, then passes through the amplification channel 308 and enters the amplification chamber 309. The constant temperature amplification reagent powder is dissolved, and the microfluidic chip is placed in a matching dedicated constant temperature fluorescence detection device to perform a DNA amplification test. The constant temperature nucleic acid amplification reaction conditions are as follows Table 3:

[0115] Table 3 Constant temperature nucleic acid amplification reaction conditions

[0116] .

[0117] (4) In the above (3), if the reaction solution needs to be supplemented before the sample (nucleic acid template) enters the amplification chamber 309 from each amplification flow channel 308, this step can be added. Before the microfluidic chip is placed in the device, the replenishing liquid (e.g., PBS solution) is pre-stored in the replenishing tube 112 on the shell 100, and the cover 105 at the top of the replenishing tube 112 seals the replenishing tube 112. Since the upper section of the replenishing tube 112 is a corrugated tube, the replenishing liquid can be made to flow downward by compressing the corrugated tube. The end of the replenishing tube 112 is sealed with a dissolving membrane. Manually press the top of the replenishing tube 112, or set a dedicated trigger at the top position of the replenishing tube 112 of the shell corresponding to the detection device, so as to apply a downward thrust to the replenishing tube 112. The manual pressure or trigger push force causes the infusion tube to move a small distance downward along the housing infusion port 111 and the top plate infusion port 203, allowing the dissolved membrane at the end of the infusion tube 112 to be punctured by the needle 314 in the infusion chamber 311. The continued pressure or trigger push compresses the corrugated tube at the upper section of the infusion tube 112, causing the liquid in the infusion tube 112 to flow into the infusion chamber 311, then from there through the bypass channel 312, before being diverted downstream to the two extraction channels 306. Under the action of negative pressure, this supplemental liquid mixes with the sample (nucleic acid template) flowing out of the extraction channel 306, is guided by the negative pressure into the diversion chamber 307, and then enters the corresponding amplification chamber 309 through each amplification channel 308.

[0118] (5) The fluorescence detection module in the device monitors the fluorescence of each amplification chamber 309 in real time. The amount of DNA increases exponentially during the amplification process. When the amount of DNA is below the detection limit, the fluorescence beam cannot be detected and is emitted in a straight line. When the DNA is amplified to at least the detection limit, the fluorescence beam increases exponentially.

[0119] 4. Validity determination:

[0120] Two of the amplification chambers 309 are used, one as a negative control, which is a blank chamber without primers and probes added, and the test result should be negative; the other is used as a positive control, which contains a positive quality control product in the reagent dry powder and the test result should be positive; otherwise, the experiment is considered invalid.

[0121] 5. Interpretation of results:

[0122] The remaining amplification chambers 309 correspond to the six pathogens mentioned above. After the amplification reaction is completed, the detection equipment automatically reads the fluorescence intensity value of each amplification chamber 309 to determine the negative or positive result.

[0123] Example 2: Specificity test

[0124] The microfluidic chip disclosed above was used to test 10 samples. Six positive samples, numbered 1 to 6, contained nucleic acids from the six pathogen-positive strains described above. Sample 1 contained nucleic acid from pseudorabies virus, sample 2 contained nucleic acid from porcine reproductive and respiratory syndrome virus, sample 3 contained nucleic acid from porcine epidemic diarrhea virus, sample 4 contained nucleic acid from porcine circovirus, sample 5 contained nucleic acid from classical swine fever virus, and sample 6 contained nucleic acid from swine influenza virus. Four negative samples were numbered 7 to 10. Testing was performed according to the method of Example 1 above, and the test results are shown in Table 4 below. Figure 12 shows the specificity test results of the recombinase isothermal amplification method based on the microfluidic chip for positive sample 3 of this Example.

[0125] Table 4 Specificity test results

[0126] .

[0127] The test results show that this kit has high specificity in microfluidic chip detection, only detecting the corresponding pathogens without cross-reacting with the nucleic acids of other pathogens.

[0128] Example 3: Sensitivity test

[0129] (1) The microfluidic chip was used to detect the mixed samples of 6 pathogen plasmids, and the dilution concentration was 1.0×10 5 copies / µL, 1.0×10 4 copies / µL, 1.0×10 3 copies / µL, 1.0×10 2 copies / µL, 1.0×10 1 copies / µL, 1.0×10 0 Sensitivity testing was conducted using six gradients of 100 copies / µL. Detection was performed according to the method described in Example 1. In the gradient sensitivity test, lower starting template counts resulted in higher Ct values. The test results showed that the kit of the present invention exhibited high sensitivity in the microfluidic chip, with Ct values ​​changing in a gradient pattern with decreasing concentration. The results are shown in Table 5.

[0130] Table 5 Sensitivity test results

[0131] .

[0132] The test results showed that this kit was highly sensitive in the diagnosis of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus, classical swine fever virus and swine influenza virus in the microfluidic chip, and could still be detected at a concentration of 10 copies / µL.

[0133] (2) A microfluidic chip was used to detect a random sample of one pathogen plasmid from the six pathogens. Porcine epidemic diarrhea virus (PEDV) was selected for recombinase isothermal amplification detection. The PEDV plasmid sample was serially diluted 10-fold to a copy number range of 1.0×10 6 ~1.0×10 1 Each gradient was used as a template for isothermal amplification using the kit of the present invention. The amplification results are shown in Figure 13, which demonstrates that the kit has a high sensitivity for detecting porcine epidemic diarrhea virus in a microfluidic chip.

[0134] Example 4: Repeatability test

[0135] The concentration of the PEDV plasmid sample was 1.0×10 3 The reproducibility test was carried out three times with 100 copies / µL as the amplification template. The test results are shown in Figure 14. The amplification curve morphology in the figure has good reproducibility, which shows that the kit has good reproducibility.

[0136] Example 5: Detection of clinical samples

[0137] Conventional PCR and isothermal amplification testing using the kit of Example 1 were performed on 20 samples of pigs suspected of pseudorabies virus (PrV) and classical swine fever virus (CSFV) sent to the clinic for testing. The test results are shown in Table 6. The results show that the isothermal amplification testing based on the embodiment of the present invention is more sensitive than conventional PCR and can rapidly perform detection and disease typing with high detection efficiency, which is of great significance for rapid isolation and targeted treatment.

[0138] Table 6 Comparison results of two detection methods

[0139] .

[0140] All the operations involving the above-mentioned pathological materials were completed at the China Animal Health and Epidemiology Center.

[0141] Example 6:

[0142] As shown in Figures 12 and 14 , the microfluidic chip of this embodiment differs from the microfluidic chip of Example 1 in that a normally open shutoff valve 500 is provided upstream of the amplification channel 308 in the microfluidic chip of this embodiment. As shown in Figures 12 and 15 , the shutoff valve 500 comprises a valve seat 501, a valve core 502, and a valve membrane 503. The valve seat 501 communicates with the amplification channel 308, and the valve core 502 is capable of producing micro-displacements in the vertical direction relative to the valve seat 501. The valve seat 501 is comprised of a diaphragm base 5012 and a plurality of valve seat body units 5011. The number of valve seat body units 5011 is the same as the number of amplification chambers 309. The valve membrane 503 is fixed between the valve seat body unit 5011 and the diaphragm base 5012. The valve seat body unit 5011 is adhesively fixed to the diaphragm base 5012, and the valve membrane 503 corresponds one-to-one with the valve seat body unit 5011. As shown in FIG13 , a corresponding stop valve connection hole 207 is provided on the top plate 200 , and a connection pool 316 is provided on each amplification channel 308 of the fluid plate 300 . Each connection pool 316 is communicated with the connection hole 207 , and the lower end of the valve core 502 is a dome.

[0143] The diaphragm base 5012 is provided with a valve diaphragm positioning groove. The valve diaphragm 503 is circular in shape and comprises a central portion that covers the connection reservoir 316, a fixed portion connected to the diaphragm base 5012, and a joint connecting the central portion and the fixed portion. The joint mates with the sidewall of the stop valve connection hole 207 on the top plate 200. The bottom of the diaphragm base 5012 is fixed to the top plate 200 and can be bonded to the top plate 200 using acrylic structural adhesive.

[0144] A valve cap 504 is mounted on the upper end of the valve core 502. The upper portion of the valve core 502 is externally threaded, while the valve seat body 5011 is internally threaded. The valve cap 504 is screwed to bring the lower end of the valve core 502 into contact with the middle portion of the valve diaphragm 503, thereby pushing the middle portion toward the connection well 316. The lower circular cross-sectional diameter of the valve core 502 is smaller than the diameter of the connection well 316. The lower circumferential surface of the valve core 502 presses the junction of the valve diaphragm 503 and the wall of the connection well 316. The valve diaphragm 503 and valve core 502 are separate components. This provides a double seal when the stop valve 500 is not closed, enhancing the sealing effect. A break wire can be designed at the junction of the fixed portion and the junction. When the middle portion of the valve diaphragm 503 is subjected to downward force, the junction separates from the fixed portion at the break wire. This ensures the integrity of the middle portion and junction of the valve diaphragm 503 and enhances the seal between the valve core 502 and the connection well 316. When the valve membrane 503 is provided with a break line, the joint part of the valve membrane 503 is separated from the fixed part after the stop valve 500 is closed, and the stop valve 500 cannot be reopened; when the valve membrane 503 is not provided with a break line, the valve membrane 503 is only elastically deformed and remains intact after the stop valve 500 is closed, and the stop valve 500 can be reopened.

[0145] In this embodiment, there are two groups of stop valves 500, each group corresponding to the amplification chamber group on that side. As shown in Figure 14, each group of stop valves 500 includes four valve seat body units 501. To prevent accidental activation of the stop valves 500, a safety clip 505 is provided between the upper end of the valve seat body unit 5011 and the valve cap 504. As shown in Figure 16, the safety clip 505 is U-shaped, and its height matches the safe distance between the upper end of the valve seat body unit 5011 and the valve cap 504. At this height, the lower end of the valve core 502 does not contact the middle portion of the valve membrane 503. The U-shaped opening of the safety clip 505 has an inwardly contracted C-shaped portion to prevent accidental detachment of the safety clip 505.

[0146] After the sample (nucleic acid template) enters the amplification flow channel 308 at a pre-set time, the shutoff valve 500 is normally open. The safety clip 505 is removed, and the valve caps 504 are manually screwed one by one to move the corresponding valve cores 502 downward, closing the amplification flow channel 308 via the valve cores 502 and valve membranes 503. Of course, when dedicated equipment is not used, a mechanical thrust can be manually applied to the shutoff valve 500 using a dedicated tool. Once closed, the shutoff valve 500 completely blocks the fluid passage. The shutoff valve 500 serves to protect the device in the event of a power failure and prevents the liquid in the amplification chamber 309 from backflowing due to other unexpected factors, causing crosstalk.

[0147] Example 7:

[0148] Compared with the microfluidic chip in Example 6, the present embodiment differs in that, as shown in FIG17 , the upper portion of the valve core 502 of the stop valve 500 in the present embodiment is not connected to the valve seat body unit 5011 by threaded connection, but is connected by a press-on connection. Pressing to close the stop valve 500 is easier and quicker to operate. The upper portion of the valve core 502 fits tightly against the inner wall of the valve seat body unit 5011, and the two can slide relative to each other under the action of external force. A plurality of flaps 506 are provided on the circumference of the lower portion of the valve core 502. The flaps 506 are based on the lower portion of the valve core 502 as a matrix. The upper end of the flap 506 is away from the lower portion of the valve core 502 as a matrix and is not connected to the matrix. The lower end of the flap 506 is integrated with the lower portion of the valve core 502. The valve seat body unit 5011 has a circle of ridges 507 on its inner wall near the lower end, which exerts a downward force on the valve cap 504, and the valve core 502 moves downward a small distance along the valve seat body unit 5011. During this process, the end of the flap 506 will first contact the ridge 507 and be squeezed by it, and the upper end of the flap 506 gradually approaches the mother body until the upper end of the flap 506 is lower than the ridge 507, and the flap 507 reopens. The upper end of the flap 507 returns to a state away from the valve core 502, and the flap 506 is in an open state. The upper end of the flap 506 abuts against the lower edge of the ridge 507 and is stuck. At this time, the lower end and side surface of the valve core 502 squeeze the valve membrane 503, and the valve core 502 and the connection pool 316 form a closed space, which can completely cut off the fluid channel. Once the stop valve 500 is closed, it cannot be reopened.

[0149] After the sample (nucleic acid template) has entered the amplification flow channel 308 for a predetermined period of time, the safety clip 505 is removed, and the valve caps 504 are manually pressed one by one to move the corresponding valve cores 502 downward, closing the amplification flow channel 308 via the valve cores 502 and valve membranes 503. Alternatively, a dedicated trigger device can be installed at the upper end of the corresponding valve caps 504 on the testing device to uniformly apply pressure to each valve cap 504 to automatically close the shutoff valve 500.

[0150] Example 8:

[0151] As shown in Figures 18 and 20 , the difference between this embodiment and the microfluidic chip in Example 1 is that the microfluidic chip in this embodiment is provided with a normally closed shutoff valve 600 in the downstream section of the extraction channel 306. This shutoff valve 600 is similar in structure to the stop valve 500 in Example 6, but in this embodiment, the lower end of the shutoff valve core 602 of the shutoff valve 600 is covered by a shutoff valve membrane 603, which can move in conjunction with the lower end of the shutoff valve core 602.

[0152] As shown in Figure 19, the bottom of the shutoff valve seat 601 of the shutoff valve 600 of this embodiment is fixed to the top plate 200, which can be bonded using acrylic structural adhesive. A shutoff valve communication hole 208 is provided on the top plate 200, and a corresponding shutoff reservoir 317 is provided downstream of the extraction channel 306 of the fluid plate 300. A shutoff valve cap 604 is provided at the upper end of the shutoff valve core 602. The upper portion of the shutoff valve core 602 is provided with external threads, and the shutoff valve seat 602 is provided with internal threads. By screwing the shutoff valve cap 604, the lower end of the shutoff valve core 602 drives the shutoff valve diaphragm 603 toward the shutoff reservoir 317. The diameter of the lower circular cross-section of the shutoff valve core 602 is smaller than the diameter of the shutoff reservoir 317. The lower circumferential side surface and lower end surface of the shutoff valve core 602 squeeze the shutoff valve diaphragm 603 into close contact with the wall of the shutoff reservoir 317.

[0153] The shutoff valve 600 remains closed, and the chip is pre-treated with negative pressure from the negative pressure inlet 108 of the chip housing, so that the pressure of the chip shunt chamber 307, amplification flow channel 308, and amplification chamber 309 is lower than the external atmospheric pressure. In this way, no external power source is required during use. Referring to Figures 5 and 6, since the microfluidic matrix on the chip has been processed, the sample flow channel 302 and buffer flow channel 304 located upstream of the extraction flow channel 306 generate adsorption force on the microfluid. The vents 310 and overflow holes 3054 ventilate the pressure in the fluid plate 300 to atmospheric pressure, ensuring that the sample reagent added from the injection chamber 301 can flow normally to the extraction chamber 305. After the sample is added, the tube cover 104 is closed and the shutoff valve 600 is opened again when it is evaluated. The reagent dry powder in the extraction chamber 305 is fully redissolved, and the pressure in the shunt chamber 307 and amplification flow channel 308 is low. The existence of the pressure difference will guide the liquid through the serpentine extraction flow channel 306, efficiently mixing, and obtaining a nucleic acid template for pathogen detection. The sample (nucleic acid template) flows into the two diversion chambers 307, then passes through the amplification channel 308 and enters the amplification chamber 309. The constant temperature amplification reagent powder is dissolved, and the microfluidic chip is placed in a matching dedicated constant temperature fluorescence detection equipment to perform a DNA amplification test.

Claims

1. A microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification, comprising a microfluidic chip, characterized in that: The microfluidic chip is provided with a sample injection chamber, an amplification chamber, and a microfluidic channel for connecting the sample injection chamber and the amplification chamber. There are at least six amplification chambers, each of which is independent of each other, and corresponding thermostatic amplification reagent dry powders are preset in each amplification chamber. The thermostatic amplification reagent dry powders preset in the amplification chambers can respectively amplify any one of the following six pathogens, and the pathogens amplified by the thermostatic amplification reagent dry powders in all the amplification chambers can cover the following six pathogens: Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, Porcine Circovirus, Classical Swine Fever Virus, and Swine Influenza Virus.

2. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 1, wherein: The thermostatic amplification reagent dry powder includes primers, probes, DNA helicase, and DNA polymerase for thermostatically amplifying the six pathogens of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, Porcine Circovirus, Classical Swine Fever Virus, and Swine Influenza Virus.

3. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 2, wherein: A buffer chamber, an extraction chamber, and a shunt chamber are also provided between the sample injection chamber and the amplification chamber of the microfluidic chip. The microfluidic channel is segmented to achieve sequential connection of the sample injection chamber, the buffer chamber, the extraction chamber, the shunt chamber, and the amplification chamber.

4. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 3, characterized in that: Corresponding nucleic acid release reagent dry powder is preset in the extraction chamber.

5. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 4, characterized in that: The outermost layer of the microfluidic chip is a housing, and inside the housing, there are a top plate, a fluid plate, and a bottom plate in sequence from top to bottom.

6. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 5, wherein: The microfluidic channel includes a sample splitting channel, a buffer channel, an extraction channel, and an amplification channel provided on the fluid plate. The sample injection chamber is connected to the buffer chamber through the sample splitting channel, the buffer chamber is connected to the extraction chamber through the buffer channel, the extraction chamber is connected to the shunt chamber through the extraction channel, and the shunt chamber is connected to each amplification chamber through the amplification channel.

7. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 6, characterized in that: The sample injection chamber is arranged at the middle position on one side of the fluid plate. There are two buffer chambers, and the two buffer chambers are respectively connected to both sides of the sample injection chamber through the sample splitting channel. There are two extraction chambers, and the two extraction chambers are respectively connected to the corresponding buffer chambers through the buffer channel. There are two shunt chambers, and the two shunt chambers are respectively connected to the corresponding extraction chambers through the extraction channel. Sampling ports corresponding to the sample injection chamber are respectively provided on the top plate and the housing.

8. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 7, wherein: The amplification chambers are arranged in two groups, respectively at both ends of the fluid plate opposite to the sample injection chamber. Each group of amplification chambers is arranged in sequence along the edge of the fluid plate in parallel. The amplification channels are in one-to-one correspondence with the amplification chambers, and each amplification chamber is connected to the corresponding shunt chamber through the amplification channel.

9. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to any one of claims 3 to 8, characterized in that: The extraction chamber includes an active mixing zone, a dry powder preset zone, and an overflow zone. There is a mixing step in the active mixing zone. The base surface of the dry powder preset zone is lower than the lowest mixing table surface of the active mixing zone. The overflow zone is arranged on one side of the active mixing zone and the dry powder preset zone. The base surface of the overflow zone is higher than the highest mixing table surface of the active mixing zone, and an overflow hole is opened in the overflow zone.

10. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 9, wherein: The extraction channel is a serpentine channel. The buffer channel is connected to the active mixing zone, and the extraction channel is connected to the dry powder preset zone.

11. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 10, characterized in that: A waste liquid chamber and a relief groove are opened on the bottom plate. An absorbent pad is arranged in the waste liquid chamber, and the overflow hole communicates with the waste liquid chamber; the relief groove is located below the amplification chamber.

12. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 11, wherein: The fluid plate is provided with ventilation holes for exhausting the waste liquid chamber, and a breathable water-blocking filter membrane is attached to the upper part of the ventilation holes. Ventilation openings are provided on the housing and the top plate corresponding to the ventilation holes.

13. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 12, characterized in that: An air flow channel communicating with the shunt chamber and an air chamber communicating with the air flow channel are further provided on the top plate. A negative pressure inlet is provided on the housing corresponding to the air chamber.

14. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 13, characterized in that: A liquid replenishment chamber and a bypass flow channel for replenishing liquid are provided on the fluid plate. A liquid replenishment pipe is provided on the housing, and the end of the liquid replenishment pipe communicates with the liquid replenishment chamber on the fluid plate. There are openings on the surfaces of the housing and the top plate for the end of the liquid replenishment pipe to pass through. The end of the liquid replenishment pipe is sealed with a dissolution membrane, and a cover is provided at the top of the liquid replenishment pipe. The upper section of the liquid replenishment pipe is a corrugated pipe, and the lower section is conical. The end of the liquid replenishment pipe faces the liquid replenishment chamber on the fluid plate. A needle-like object is provided at the center of the liquid replenishment chamber on the fluid plate opposite to the end of the liquid replenishment pipe. The bypass flow channel connects the liquid replenishment chamber and the extraction flow channel.

15. The microfluidic chip kit for rapid nucleic acid detection based on recombinase aided isothermal amplification according to any one of claims 10 to 14, characterized in that: A thermal isolation area is further provided on the fluid plate. The thermal isolation area includes a plurality of thermal isolation grooves respectively surrounding the amplification flow channel and the amplification chamber.

16. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 15, characterized in that: A label covering area is provided on the surface of the housing, and a label is attached to the label covering area; a sample indication area is provided on the housing, the sample indication area is located beside the sample injection chamber, and a detection opening is provided on the housing.

17. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 16, wherein: A normally open cut-off valve is provided on the amplification flow channel. The cut-off valve includes a valve seat, a valve core, and a valve membrane. The valve seat communicates with the amplification flow channel, and the valve core can generate a micro-displacement downward relative to the valve seat; the valve seat includes a diaphragm base and a plurality of valve seat body units connected together. The diaphragm base is fixed on the top plate, and the number of valve seat body units is the same as the number of amplification chambers. The valve membrane is fixed between the valve seat body units and the diaphragm base, and the valve membrane corresponds to the valve seat body units one by one. When the cut-off valve is closed, the valve core and the valve membrane cut off the amplification flow channel.

18. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 16 or 17, characterized in that: A normally closed shut-off valve is provided on the extraction flow channel. The shut-off valve includes a shut-off valve seat, a shut-off valve core, and a shut-off valve membrane. The shut-off valve seat communicates with the extraction flow channel, and the shut-off valve core can generate a micro-displacement downward relative to the shut-off valve seat; the shut-off base is fixed on the top plate, and the shut-off valve membrane and the lower end of the shut-off valve core are coated as one body. When the shut-off valve is opened, the extraction flow channel communicates with the shunt chamber.

19. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 2, characterized in that: The sequences of the primers are: Porcine epidemic diarrhea virus, SEQ ID NO.1-2; Porcine reproductive and respiratory syndrome virus, SEQ ID NO.4-5; Porcine pseudorabies virus, SEQ ID NO.7-8; Porcine circovirus, SEQ ID NO.10-11; Classical swine fever virus, SEQ ID NO.13-14; Porcine influenza virus, SEQ ID NO.16-17; The sequences of the probes are: Porcine epidemic diarrhea virus, SEQ ID NO.3; Porcine reproductive and respiratory syndrome virus, SEQ ID NO.6; Porcine pseudorabies virus, SEQ ID NO.9; Porcine circovirus, SEQ ID NO.12; Classical swine fever virus, SEQ ID NO.15; Porcine influenza virus, SEQ ID NO.

18.

20. The microfluidic chip kit for rapid nucleic acid detection based on recombinase polymerase amplification according to claim 19, wherein: Two of the amplification chambers on the microfluidic chip are set as a positive control chamber and a negative control chamber. The positive control chamber is pre-loaded with a positive control product, and the positive control product contains plasmids corresponding to the amplification gene sequences of the above six pathogens.

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