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

Abstract

The invention discloses a microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification, wherein the microfluidic chip is provided with a sample injection chamber, a buffer chamber, an extraction chamber, a shunt chamber, an amplification chamber, and a microfluidic flow channel for realizing sequential communication of each chamber; the extraction chamber is pre-installed with a corresponding nucleic acid release reagent dry powder; each amplification chamber is pre-installed with a corresponding isothermal amplification reagent dry powder, and the isothermal amplification reagent dry powder pre-installed in the amplification chamber can respectively amplify any one of the six common pathogens in pigs, and the pathogens amplified by the isothermal amplification reagent dry powder in all amplification chambers can include six pathogens. Nucleic acid extraction and amplification reaction are integrated on one chip, and the detection requirements of rapid detection of multiple pathogens at the same time are realized, with good sensitivity and specificity, and the operation process is also simplified, less time-consuming, and the detection cost is reduced.

Description

Technical field:

[0001] 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 technology:

[0002] In recent years, the rapid development of animal husbandry has become increasingly large-scale and commercialized, but the occurrence of various animal diseases in the breeding process has always been a major hidden danger in animal husbandry production. Among them, animal infectious diseases are extremely harmful diseases caused by various pathogens. They will spread among livestock and spread very quickly. They are animal plagues with certain clinical symptoms and incubation periods. For example:

[0003] Porcine epidemic diarrhea is an important acute, contact, viral intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). PEDV can persist in pigs and pigs of all ages are susceptible. Therefore, once the pigs are infected, it will cause huge economic losses to farmers.

[0004] 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 my country's pig industry.

[0005] 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.

[0006] 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.

[0007] Swine flu is an acute respiratory disease caused by swine influenza virus (SIV), which not only seriously threatens the development of my country's pig industry, but also endangers human health.

[0008] Therefore, early detection of the nucleic acid of the above pathogens can not only understand the infection situation of the pig farm, take measures early to control the epidemic, but also help the positive farms resume production. In terms of detecting the nucleic acid of the above pathogens, the most widely used one is the polymerase chain reaction (PCR), but PCR requires expensive instruments and equipment, skilled technicians, and a long detection time. It is difficult to promote it at the grassroots level or in resource-scarce areas, and it cannot be used for on-site detection outside the laboratory. The nucleic acid constant temperature amplification technology that has been developed simplifies the amplification conditions and does not require a thermal cycler, which greatly reduces the dependence on complex instruments. As an important member of the nucleic acid constant temperature amplification, the recombinase polymerase amplification technology is simpler to operate than other constant temperature amplification technologies. It does not require initial heating and does not require repeated enzyme addition during the reaction cycle. It is very suitable for early clinical rapid diagnosis and on-site real-time detection. On-site real-time detection of clinical samples is also one of the effective means to block the rapid spread of the disease. At the same time, fast and reliable diagnostic tools help to better implement the correct treatment methods.

[0009] 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, the 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 basically integrate basic operating units such as sample preparation, reaction, separation, and detection. The microchannel forms a network and runs through the entire system with controllable fluids. The sample consumption is small, the detection is fast, and closed automated detection is easy to achieve.

[0010] 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 optimize the isothermal amplification technology in combination with microfluidic chips 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 clinically suspected mixed infections or cases with similar clinical manifestations, rapid screening or disease typing cannot be achieved. In particular, the detection of multiple pathogens requires different detection kits to be used in combination, 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 to quickly detect multiple pathogens at the same time, and can reduce the complexity of integrated nucleic acid extraction and amplification, meet the testing and diagnosis needs related to nucleic acids, and have important practical significance for achieving simple and efficient nucleic acid diagnostic analysis.

[0011] 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:

[0012] The present invention provides a microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification. Nucleic acid extraction and amplification reactions are integrated on one chip to achieve the requirement of rapid and simultaneous detection of multiple pathogens. The kit has good sensitivity and specificity. In addition, the operation process is simplified, less time is consumed, and the detection cost is reduced.

[0013] The technical solution adopted by the present invention to solve the above technical problems is:

[0014] 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 a sample injection chamber, a buffer chamber, an extraction chamber, a shunt chamber, an amplification chamber, and a microfluidic flow channel for sequentially connecting the sample injection chamber, the buffer chamber, the extraction chamber, the shunt chamber, and the amplification chamber; the extraction chamber is pre-installed with a corresponding nucleic acid release reagent dry powder; the amplification chamber comprises at least six amplification chambers, each of which is independent of each other, and each amplification chamber is pre-installed with a corresponding isothermal amplification reagent dry powder, the isothermal amplification reagent dry powder pre-installed in the amplification chamber can respectively amplify any one of the following six pathogens, and the pathogens amplified by the isothermal amplification reagent dry 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; the isothermal amplification reagent dry powder comprises primers, probes, DNA helicase and DNA polymerase for isothermal amplification of the above six pathogens.

[0015] As an improved solution of the present invention, the outermost layer of the microfluidic chip is a shell, and the shell is composed of a top plate, a fluid plate and a bottom plate from top to bottom; the microfluidic flow channel includes a sample splitting flow channel, a buffer flow channel, an extraction flow channel and an amplification flow channel, and the injection chamber is provided in the middle position of one side of the fluid plate, the two buffer chambers are connected to the injection chamber through the sample splitting flow channel, the two extraction chambers are connected to the corresponding buffer chambers through the buffer flow channel, and the two shunt chambers are connected to the corresponding extraction chambers through the extraction flow channel, and the top plate and the shell are respectively provided with injection ports corresponding to the injection chambers; the amplification chambers are arranged in two groups, which are respectively located at the two ends of the other side of the fluid plate, and each group of amplification chambers is arranged in sequence along the end of the fluid plate parallel to the amplification flow channel, the amplification flow channel is connected to the amplification chamber one by one, and each amplification chamber is connected to the corresponding shunt chamber through the amplification flow channel.

[0016] 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, the extraction flow channel is a serpentine flow channel, the buffer flow channel is connected to the active mixing zone, and the extraction flow channel is connected to the dry powder pre-setting zone.

[0017] As a further improvement of the present invention, a waste liquid chamber and a clearance groove are provided on the bottom plate, an absorption pad is arranged 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.

[0018] 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.

[0019] As a further improvement of the present invention, an air flow channel communicating with the diversion chamber and an air chamber communicating with the air flow channel are further provided on the top plate, and a negative pressure inlet is provided on the shell corresponding to the air chamber.

[0020] As a further improvement of the present invention, a fluid infusion chamber and a bypass flow channel for replenishing liquid are provided on the fluid plate, a fluid infusion tube is provided on the shell, the end of the fluid infusion tube is communicated with the fluid infusion chamber on the fluid plate, and openings for the end of the fluid infusion tube to pass through are provided on the shell and the surface of the top plate. The end of the fluid infusion tube is sealed with a dissolving membrane, and a sealing cover is provided on 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 in the fluid infusion chamber of the fluid plate at the center of the end, and the bypass flow channel connects the fluid infusion chamber and the two extraction flow channels.

[0021] As a further improvement of the present invention, the fluid plate is further provided with a heat isolation area, the heat isolation area includes a plurality of heat isolation grooves, and the plurality of heat isolation grooves surround the amplification flow channel and the amplification chamber respectively. A label covering area is provided on the surface of the shell, and a label is attached to the label covering area; a sample indication area is provided on the shell, and the sample indication area is located next to the injection chamber, and a detection port is provided on the shell.

[0022] As a further improvement of the present invention, a normally open stop valve is arranged 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 a plurality of valve seat body units connected together, the diaphragm base is fixed on the top plate, the number of the 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, the valve membrane corresponds to the valve seat body unit one by one, and when the stop valve is closed, the valve core and the valve membrane cut off the amplification flow channel.

[0023] As a further improvement of the present invention, a normally closed shut-off valve is arranged 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 is integrally wrapped with the lower end of the shut-off valve core. When the shut-off valve is opened, the extraction flow channel is communicated with the diversion chamber.

[0024] 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.

[0025] As a further improvement of the present invention, two of the amplification chambers on the microfluidic chip are configured 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 a plasmid corresponding to the amplified gene sequences of the above six pathogens.

[0026] Beneficial effects of the present invention:

[0027] The microfluidic chip kit provided by the present invention can make a liquid sample flow through each chamber in an orderly manner, independently of each other, and without interfering with each other, through a sample injection chamber, a buffer chamber, an extraction chamber, a shunt chamber and an amplification chamber, and a microfluidic flow channel that realizes sequential communication between the sample injection chamber, the buffer chamber, the extraction chamber, the shunt chamber and the amplification chamber.

[0028] The kit of the present invention combines the constant temperature amplification technology with the microfluidic chip to simultaneously detect six pathogens that the pig herd is susceptible to; it has strong anti-interference performance, and a good amplification result 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, consuming less time, and having low technical requirements for operators; it avoids sample contamination that may be caused during the experiment, and the detection results are stable and reliable, with good reproducibility, strong specificity, and high sensitivity.

[0029] The microfluidic chip of the present invention integrates nucleic acid extraction and DNA amplification. The detection process is a closed reaction, which reduces the experimental steps, reduces the risk of contamination and the possibility of result deviation. The entire system does not contain harmful substances, and the amplification product does not need special post-treatment.

[0030] The structural setting of the extraction chamber in the microfluidic chip of the present invention makes the reaction mixing efficient, the rehydration chamber can replenish the reaction reagent in time, and the dry powder reagent is fully redissolved to ensure that the amount of mixed liquid during amplification is sufficient for stable amplification. The setting of the waste liquid chamber ensures that there is no fluid leakage during detection, which is harmless to the operator and the environment; the addition of the stop valve ensures that there is no leakage risk even if the chip is inverted or fails, and the safety is high, while preventing interference between amplification chambers; the addition of the interception valve enables the chip to maintain a negative pressure state in advance, without the need to introduce an external drive, reducing the complexity of the equipment, and facilitating real-time detection. Description of the drawings:

[0031] Figure 1 The figure is a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention.

[0032] Figure 2 The figure is a schematic structural diagram of a shell according to an embodiment of the present invention.

[0033] Figure 3 A schematic diagram of a microfluidic chip without a housing structure according to an embodiment of the present invention.

[0034] Figure 4 The top plate structure schematic diagram of an embodiment of the present invention.

[0035] Figure 5 This is a schematic diagram of the fluid plate structure of an embodiment of the present invention.

[0036] Figure 6 This is a front view of a fluid plate according to an embodiment of the present invention.

[0037] Figure 7 The figure is a schematic structural diagram of a base plate according to an embodiment of the present invention.

[0038] Figure 8 This is a simplified indication diagram of the functions of the microfluidic chip according to an embodiment of the present invention.

[0039] Fig. 9 This is a graph showing the specificity test results of the recombinase isothermal amplification method based on a microfluidic chip.

[0040] Fig.10 This is a sensitivity test result of the recombinase isothermal amplification method based on a microfluidic chip.

[0041] Fig.11 This is a graph showing the repeatability test results of the recombinase isothermal amplification method based on a microfluidic chip.

[0042] Fig.12 FIG. 1 is a schematic diagram of a microfluidic chip without a housing structure according to another embodiment of the present invention.

[0043] Fig.13 This is a schematic diagram of the top plate structure of another embodiment of the present invention.

[0044] Fig.14 FIG. 4 is a schematic diagram of the structure of a microfluidic chip according to another embodiment of the present invention.

[0045] Fig.15 It is a cross-sectional view of the first stop valve structure in another embodiment of the present invention, wherein Fig.15 a is the stop valve is not closed, Fig.15 b is the stop valve closed state.

[0046] Fig.16 This is a schematic diagram of the structure of a first stop valve in another embodiment of the present invention.

[0047] Fig.17 FIG. 1 is a cross-sectional view of a second stop valve structure in another embodiment of the present invention, wherein Fig.17 a is the stop valve is not closed, Fig.17 b is the stop valve closed state.

[0048] Fig.18 This is a schematic diagram of a microfluidic chip without a housing structure according to another embodiment of the present invention.

[0049] Fig.19 The figure is a cross-sectional view of the shut-off valve structure in another embodiment of the present invention.

[0050] Fig. 20 The figure is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.

[0051] Figure 1 In the figure, 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;

[0052] Figure 2 In the figure, 100. shell, 101. detection port, 102. shell vent, 103. sealant plug port, 104. tube cover, 105. cover, 106. label covering area, 107. sample indication area, 108. shell negative pressure inlet, 109. wing, 110. shell injection port, 111. shell refill port, 112. refill tube;

[0053] Figure 3 In the middle, 200. top plate, 300. fluid plate, 400. bottom plate;

[0054] Figure 4 In the figure, 201. top plate sample inlet, 202. top plate vent, 203. top plate liquid replenishing port, 204. air flow channel, 205. air chamber, 206 top plate assembly hole;

[0055] Figure 5 and Figure 6 In the figure, 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. flow distribution 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;

[0056] Figure 7 In the middle, 400. bottom plate, 401. waste liquid chamber, 402. groove, 403. flat support, 404. column, 405. give way slot;

[0057] Figure 8 In the middle, 204. air flow channel, 205. air chamber, 301. injection chamber, 302. sample distribution channel, 303. buffer chamber, 304. buffer channel, 305. extraction chamber, 306. extraction channel, 307. flow distribution chamber, 308. amplification channel, 309. amplification chamber, 311. liquid replenishment chamber, 312. bypass channel, 313. thermal isolation tank, 401. waste liquid chamber;

[0058] Fig.12 In the middle, 200. top plate, 300. fluid plate, 400. bottom plate, 500. stop valve;

[0059] Fig.13 Among them, 201. top plate sample inlet, 202. top plate vent, 203. top plate liquid replenishing port, 204. air flow channel, 205. air chamber, 206 top plate assembly hole, 207. stop valve connection hole;

[0060] Fig.14 In the figure, 100. housing, 101. detection 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;

[0061] Fig.15 a and Fig.15 b, 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;

[0062] Fig.16 In the figure, 5011. valve seat body unit, 5012. base, 504. valve cap, 505. safety buckle;

[0063] Fig.17 a and Fig.17 In b, 501. valve seat, 5011. valve seat body unit, 5012. diaphragm base, 502. valve core, 503. valve membrane, 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;

[0064] Fig.18 In the middle, 200. top plate, 300. fluid plate, 400. bottom plate, 600. shut-off valve;

[0065] Fig.19 In the figure, 601. shutoff valve seat, 602. shutoff valve core, 603. shutoff valve membrane, 604. shutoff valve cap, 200. top plate, 2087. shutoff valve communication hole, 300. fluid plate, 308. amplification flow channel, 317. shutoff pool;

[0066] Fig. 20 In the figure, 100. shell, 101. detection 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. Specific implementation method:

[0067] The present invention combines the constant temperature amplification technology with the microfluidic chip technology. Now, a brief description of the constant temperature amplification technology is given. Body Temperature Nucleic Acid Amplification (BTNAA) is a technology in which the recombinase and primer form a protein / single-stranded nucleotide complex Rec / ssDNA at a constant temperature, and invade the double-stranded DNA template with the help of auxiliary protein and single-stranded binding protein SSB; a D-loop region is formed at the invasion site, and the DNA double strand is scanned; after finding the target region complementary to the primer, the complex Rec / ssDNA disintegrates, and the polymerase also binds to the 3' end of the primer to start chain extension. This recombinase polymerase constant temperature amplification technology has unique application advantages as a rapid, convenient and specific diagnostic method.

[0068] In order to better describe the present embodiment, the following explanation is now made: the upstream or downstream of the flow channel mentioned below refers to the section of the flow channel through which the liquid first flows as the upstream, and the section through which the liquid flows later as the downstream. Since the microfluidic chip sample requires a small amount 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 such as "inside", "outside", "left", "right", "up", "down", "side", "bottom", "front", "back" and the like mentioned below indicate the orientation or position relationship based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the structural relationship of each component or element in the present invention. The relationship words determined cannot be understood as limiting the present invention. The parts not described in detail in the present invention are all known technologies of technicians in this technical field.

[0069] Embodiment 1:

[0070] like Figure 1 , Figure 3 and Figure 5 As shown, the microfluidic chip kit for rapid detection of nucleic acid 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 arranged, as well as a microfluidic 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. Wherein: the extraction chamber 305 is pre-installed with corresponding nucleic acid release reagent dry powder, the main components of which are lithium dodecyl sulfate and NaCl; the six amplification chambers 309 are pre-installed with corresponding constant temperature amplification reagent dry powder, and the constant temperature amplification reagent dry powder pre-installed in the amplification chambers 309 can respectively amplify any of the following six pathogens, and at the same time, the pathogens amplified by the constant temperature amplification reagent dry 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 dry powder in the present invention includes primers, probes, DNA helicase and DNA polymerase for constant temperature amplification of the above six pathogens.

[0071] 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.

[0072] 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.

[0073] There are two amplification chambers 309 on the microfluidic chip, which are set as a positive control chamber and a negative control chamber. The 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, which contains a plasmid corresponding to the amplified gene sequences of the above six pathogens. The negative control chamber is a blank chamber. Figure 3 As shown, due to the addition of a positive control chamber and a negative control chamber, a total of eight amplification chambers 309 are arranged on the microfluidic chip, and each amplification chamber 309 is independent of each other.

[0074] The microfluidic chip according to the embodiment of the present invention will be described in detail below.

[0075] like Figures 1 to 3As shown, the uppermost 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, and 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, and the bottoms of the two wings 109 of the housing 100 are embedded in the grooves 402 on the bottom plate 400, and the two inner sides of the housing 100 can resist 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, and a detection port 101 is also provided on the housing 100, and the optical module of the matching constant temperature fluorescence detection device can emit excitation light from the detection port 101 into the product to be detected and converge on the product to be detected, so as to excite the product to be detected to react and produce fluorescence. The housing 100, the top plate 200 and the fluid plate 300 are all provided with assembly holes, such as the top plate assembly hole 206 on the top plate, the fluid plate assembly hole 315 on the fluid plate 300, the bottom plate 400 is provided with a column 404 for limiting the assembly hole, and the bottom plate 400 is also provided with a flat support 403 in contact with the top plate 200, and the flat support 403 can also limit the position of the fluid plate 300. The right side of the surface of the housing 100 is provided with a label covering area 106, and the label covering area 106 is attached with a label. The label can have a machine-readable QR code or barcode. After scanning, the computer can automatically read and identify, obtain and / or determine the test number, test name, batch number and shelf life of the chip, etc. In order to facilitate fluorescence detection and liquid display of the sample indication area 107 on the fluid plate 300, the top plate 200, the fluid plate 300 and the 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 protect the important main body of the chip and facilitate stable holding in the detection equipment.

[0076] like Figure 5 and Figure 6As shown, the injection chamber 301 is arranged at the middle position of the front side of the fluid plate 300. The buffer chambers 303 are respectively arranged 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 for the first time when the sample is appropriate, and can also ensure that the subsequent reaction chambers are filled with sufficient amount when the sample is excessive. Even if the speed of adding the sample is fast, the two buffer chambers 303 can first drain and store the sample reagents in the injection chamber 301, and the sample reagents can also be evenly distributed to the two buffer chambers 303. The extraction chamber 305 includes an active mixing zone 3051, a dry powder pre-setting zone 3052 and an overflow zone 3053. The active mixing zone 3051 is provided with a plurality of mixing steps. The base surface of the dry powder pre-setting zone 3052 is lower than the lowest mixing table surface of the active mixing zone 3051. The overflow zone 3053 is arranged on one side of the active mixing zone 3051 and the dry powder pre-setting zone 3052. The base surface of the overflow zone 3053 is higher than the highest mixing table surface of the active mixing zone 3051. The lowest level of the plurality of mixing steps is slightly inclined toward the overflow zone 3053 side, and the two sides of each level are tangent to the side wall of the active mixing zone 3051. The dry powder pre-placement area 3052 is located at the convex angle downstream of the extraction chamber 305, and the convex angle protrudes toward the rear side of the chip. The two sides of the dry powder pre-placement area 3052 are tangentially connected to the active mixing area 3051. The nucleic acid release reagent dry powder is fixed in the dry powder pre-placement area 3052. The fixing method can be to heat and melt the paraffin and spray it on the surface of the reagent dry powder, and then fix the reagent dry powder in the dry powder pre-placement area 3052 after cooling and solidification. In this embodiment, the active mixing area 3051 is preferably a cylindrical groove structure, and the step table profile curve of each mixing step in the active mixing area 3051 is a fitting arc curve. The overflow hole 3054 is provided in the overflow area 3053, the buffer flow channel 304 is connected to the active mixing area 3051, and the extraction flow channel 306 is connected to the dry powder pre-placement area 3052. When the sample reagent enters the extraction chamber 305, it first passes through the active mixing area 3051 for mixing before flowing to the dry powder pre-placement area 3052. The active mixing flow area 3051 destroys the stratified flow, and by introducing disturbances, enhances the mixing between the flow layers, increases the probability of contact between the fluids, and thus obtains good mixing with the reagent dry powder. After fixing the reagent dry powder, it is also possible to consider spraying paraffin in the fluid confluence area of ​​the dry powder pre-setting area 3052 and wait for it to solidify to form a hydrophobic wall to form a barrier screen to increase the reaction time of the sample reagent in the dry powder pre-setting area 3052. The hydrophobic wall cannot completely prevent the reagent from flowing from the outlet of the dry powder pre-setting area 3052 to the extraction flow channel 306.

[0077] After nucleic acid extraction from the sample, before the amplification reaction, the mixed liquid needs to be evenly distributed to each amplification flow channel 308 leading to the amplification chamber 309. In order to fill all the amplification flow channels 308 with liquid, the mixed liquid needs to be diverted through the diversion chamber 307. The diversion chamber 307 is a long and narrow strip, and the amplification chambers 309 are arranged in two groups. Each amplification chamber 309 of each group is connected to a diversion chamber 307. The two groups of amplification chambers 309 are respectively located at the ends on the left and right sides of the fluid plate 300, and each group of amplification chambers 309 is arranged in sequence parallel to the left and right ends of the fluid plate 300.

[0078] like Figure 5 and Figure 6 As shown, 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 injection chamber 301 through the sample splitting channel 302, the two extraction chambers 305 are connected to the corresponding buffer chambers 303 through the buffer channel 304, the two diversion chambers 307 are connected to the corresponding extraction chambers 305 through the extraction channel 306, the amplification channel 308 is connected to the diversion chamber 307 upstream, and the amplification channel 308 is connected to the amplification chamber 309 downstream in a one-to-one correspondence.

[0079] like Figure 2 and Figure 4 As shown, the top plate 200 is provided with a top plate inlet 201 corresponding to the inlet chamber 301, and the shell 100 is provided with a shell inlet 110 corresponding to the inlet chamber 301. The extraction flow channel 306 is a serpentine flow channel. The extraction flow channel 306 in this embodiment is a combination of a semicircular flow channel and a straight flow channel. The mixing in a short time is achieved by changing the geometric shape of the flow channel. When the mixed liquid flows through the semicircular flow channel, the flow direction changes, the fusion between the liquids is enhanced, and the mixing in the span direction is enhanced.

[0080] like Figure 7 As shown, in this embodiment, each group of amplification chambers 309 has four, and directly below each group of amplification chambers 309 is a clearance groove 405 of the bottom plate 400, and each group of four amplification chambers 309 is arranged in sequence along the end edge of the chip to ensure that each amplification chamber 309 is independent of each other and separated from each other to prevent cross contamination. For reference, the clearance groove 405 can provide a heat transfer area for the heating module of the supporting constant temperature fluorescence detection equipment to heat the amplification chamber 309; it can also provide an optical path collection area for the fluorescence detection module.

[0081] like Figure 1 As shown, the sample indication area 107 on the housing 100 is located next to the injection chamber. Figure 3 and Figure 4As shown, the top plate 200 is also provided with an air passage 204 communicating with the flow dividing chamber 307, an air chamber 205 is provided on the top plate 200 corresponding to the air passage 204, and a shell negative pressure inlet 108 is provided on the shell 100 corresponding to the air chamber 205, wherein the shell negative pressure inlet 108 on the shell 100 is adapted to the sealing rubber plug 103. The shell sample inlet 110 is adapted to the tube cover 104.

[0082] like Figure 7 As shown, the outer edge of the top of the bottom plate 400 is adhered to the bottom of the fluid plate 300, and the space between the fluid plate 300 and the bottom plate 400 is a waste liquid chamber 401. An absorbent pad is arranged in the waste liquid chamber 401, and the overflow hole 3054 of the extraction chamber 305 on the fluid plate 300 is connected to the waste liquid chamber 401. The waste liquid chamber 401 is inclined toward the middle on both sides. Figure 5 As shown, the fluid plate 300 has a ventilation hole 310 for exhausting the waste liquid chamber 401, and a breathable and water-blocking filter membrane is attached to the upper part of the ventilation hole 310; Figure 2 As shown, a shell vent 102 is provided on the shell 100 at a location corresponding to the vent 310 , and a top plate vent 202 is provided on the top plate 200 at a location corresponding to the vent 310 . A sealing film is attached to the top plate vent 202 to prevent bacteria while ensuring normal gas exchange.

[0083] like Figure 2 and Figure 3 As shown, the shell 100 is provided with a liquid infusion tube 112, and the fluid plate 300 is also provided with a liquid infusion chamber 311. The end of the liquid infusion tube 112 is connected to the liquid infusion chamber 311 on the fluid plate 300. The shell 100 and the top plate 200 have openings for the liquid infusion tube 112 to pass through, such as the shell liquid infusion port 111 and the top plate liquid infusion port 203 (see Figure 4 ), the end of the infusion tube 112 is sealed with a dissolving film, and a sealing cap 105 is provided at the top of the infusion tube 112. The upper section of the infusion tube 112 is a corrugated tube, and the lower section is conical. The end of the infusion tube 112 faces the infusion chamber 311 of the fluid plate 300, and a needle 314 is provided in the infusion chamber 311 of the fluid plate 300 at the center of the end. The infusion chamber 311 is connected to the bypass flow channel 312, and the bypass flow channel 312 is set in a Y shape to be connected to the downstream sections of the two extraction flow channels 306 respectively.

[0084] like Figure 6As shown, a heat isolation area is also provided on the fluid plate 300, and the heat isolation area includes a plurality of small heat isolation grooves 313, which surround the amplification flow channel 308 and the amplification chamber 309 respectively. A closed space is formed between the heat 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 surroundings. The still air trapped in the closed space is a poor conductor of heat and has good thermal insulation properties, so that the amplification chamber 309 is easy to maintain a constant temperature. 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.

[0085] 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.

[0086] The primer and probe sequence information is shown in Table 1 below. The modified probe sequence information is shown in Table 2 below.

[0087] Table 1 Primer and probe sequence information

[0088]

[0089]

[0090] Table 2 Primer and probe sequence information

[0091]

[0092] 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 quenching group; 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.

[0093] Kit operation and result determination:

[0094] 1. Sample Collection and Processing

[0095] Sample collection is performed as needed.

[0096] (1) Oral swab sample processing: Oscillate the oral swab tube at 6000 r / min for 45 seconds and take 200 μL for later use.

[0097] (2) Processing of spleen, lung, kidney and other tissue samples: Take 0.05 g of tissue block and put it into a grinding tube filled with PBS buffer. Grind at 6000 r / min for 45 s to make about 10% tissue homogenate. Centrifuge at 5000 g for 5 min and take 200 μL of supernatant for later use.

[0098] (3) Treatment of feces and feed samples: 1g of feces or feed was placed in a grinding tube containing PBS buffer, and the mixture was ground at 6000r / min for 45s to make a 10% homogenate. The mixture was centrifuged at 5000g for 5min, and 200μL of the supernatant was taken for later use. (PBS: phosphate buffer saline)

[0099] (4) Sewage sample treatment: Take 200 μL of sewage for later use.

[0100] (5) Non-hemolyzed serum or plasma: No pretreatment is required.

[0101] 2. Amplification reaction and detection:

[0102] (1) Open the tube cover of the shell injection port 110 on the microfluidic chip shell 100, and the sample flows into the injection chamber 301 from the shell injection port 110 and the top plate injection port 201. The sample in the injection chamber 301 flows into the sample distribution channels 302 on both sides and reaches the corresponding buffer chamber 303. The sample flow can be seen from the sample indication area 107 on the shell 100, and then flows through the sample distribution channel 302 until all the samples flow to the buffer chamber 303. The presence 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 injection port 110. Allow the sample to be excessive so that the sample volume added to the injection chamber 301 is large enough to ensure proper filling in the amplification stage.

[0103] The ventilation holes 310 on the fluid plate 300 for exhausting the waste liquid chamber 401, the top plate ventilation holes 202 on the top plate 200, and the shell ventilation holes 102 on the shell 100 maintain the pressure balance between the waste liquid chamber 401 and the external environment. The waste liquid chamber 401 also ventilates the pressure in the fluid plate 300 to atmospheric pressure through the overflow holes 3054 of the extraction chamber 305 to ensure the air pressure balance in the flow channel and avoid the negative impact of air pressure hindering the normal flow of samples in the fluid plate 300.

[0104] (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 that 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, without the need for an external power source, without increasing the complexity of the structure, and is easy to reduce the volume of the equipment, which can be well adapted to on-site timely detection. The gas in the fluid plate 300 flows out through the gas flow channel 204 and the gas chamber 205, and the pressure of the diversion chamber 307 and the amplification flow channel 308 is reduced, driving the liquid into the serpentine part of the extraction flow channel 306 and mixing, 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, and 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.

[0105] (3) The sample (nucleic acid template) flows evenly into each flow chamber 307, and then enters the amplification chamber 309 through the amplification channel 308. 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 shown in Table 2:

[0106] Table 2 Constant temperature nucleic acid amplification reaction conditions

[0107]

[0108] (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 (such as 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 flow downward by compressing the corrugated tube. The end of the replenishing tube 112 is sealed with a dissolving film. Manually press the top of the replenishing tube 112, or set a special 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 pressing force or the pushing force of the trigger is strong enough to make the infusion tube move a small distance downward along the shell infusion port 111 and the top plate infusion port 203, so that the dissolved membrane at the end of the infusion tube 112 is pierced by the needle 314 in the infusion chamber 311. The pressing force or the pushing force of the trigger continues to act, and the corrugated tube at the upper part of the infusion tube 112 is compressed, and the liquid in the infusion tube 112 flows into the infusion chamber 311, and then flows from the infusion chamber 311 through the bypass channel 312, and then is diverted to the downstream of the two extraction channels 306. Under the action of negative pressure, the supplementary liquid is mixed with the sample (nucleic acid template) flowing out of the extraction channel 306, and is guided into the diversion chamber 307 by negative pressure, and then enters the corresponding amplification chamber 309 through each amplification channel 308.

[0109] (5) The fluorescence detection module in the device performs real-time fluorescence monitoring of each amplification chamber 309. The amount of DNA in the amplification process increases exponentially. When the amount of DNA is lower than the detection limit, the fluorescence beam cannot be detected and it is emitted in a straight line. When the DNA is amplified to at least the detection limit, the fluorescence beam increases exponentially.

[0110] 4. Validity determination:

[0111] Two of the amplification chambers 309, one is used as a negative control, the negative control amplification chamber is a blank chamber, no primers and probes are added, and the test result should be negative; one is used as a positive control, the reagent dry powder of the positive control amplification chamber contains a positive quality control product, and the test result should be positive; otherwise the experiment is considered invalid.

[0112] 5. Interpretation of results:

[0113] The remaining amplification chambers 309 correspond to the six pathogens respectively. After the amplification reaction is completed, the detection device automatically reads the fluorescence intensity value of each amplification chamber 309 to determine the negative or positive result.

[0114] Example 2: Specificity test

[0115] The microfluidic chip disclosed above was used to detect 10 samples, and 6 positive samples contained nucleic acids of the above 6 pathogen positive strains, numbered 1 to 6, among which sample No. 1 contained pseudorabies virus nucleic acid, sample No. 2 contained porcine reproductive and respiratory syndrome virus nucleic acid, sample No. 3 contained porcine epidemic diarrhea virus nucleic acid, sample No. 4 contained porcine circovirus nucleic acid, sample No. 5 contained classical swine fever virus nucleic acid, and sample No. 6 contained swine influenza virus nucleic acid. 4 negative samples were numbered 7 to 10. The detection operation was performed according to the method of Example 1 above, and the detection results are shown in the following Table 3. Among them, Fig.12 This is a graph showing the specificity test results of the recombinase isothermal amplification method based on a microfluidic chip for the positive sample No. 3 in this example.

[0116] Table 3 Specificity test results

[0117]

[0118] The test results show that this kit has high specificity in microfluidic chip detection, and only detects the corresponding pathogens without cross-reacting with the nucleic acids of other pathogens.

[0119] Example 3: Sensitivity test

[0120] (1) The microfluidic chip was used to detect the mixed samples of 6 pathogen plasmids, and the samples were diluted to a concentration of 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 The sensitivity test was performed with six gradients of 100 copies / μL. The detection was performed according to the method of Example 1 above. In the gradient sensitivity test, the lower the starting number of the template, the greater the Ct value. The test results show that the kit of the present invention has a high sensitivity in the microfluidic chip, and the Ct value changes in a gradient as the concentration decreases. The results are shown in Table 4.

[0121] Table 4 Sensitivity test results

[0122]

[0123] The test results show that this kit is 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 can still be detected at a concentration of 10 copies / μL.

[0124] (2) A random sample of one pathogen plasmid was detected using a microfluidic chip. Porcine epidemic diarrhea virus (PEDV) was selected for isothermal amplification of the recombinase. The PEDV plasmid sample was diluted 10 times in a gradient series to a copy number range of 1.0×10 6 ~1.0×10 1 Each gradient was used as a template and the kit of the present invention was used to perform isothermal amplification of the recombinase. The amplification results are shown in Fig.13 ,The results showed that this kit has a high detection sensitivity for porcine epidemic diarrhea virus in microfluidic chips.

[0125] Example 4: Repeatability test

[0126] The concentration of PEDV plasmid sample was 1.0×10 3 copies / μL, and used as amplification template for 3 repetitive tests. The test results were as follows Fig.14 As shown in the figure, the amplification curve has good reproducibility, and the results show that this kit has good reproducibility.

[0127] Example 5: Detection of clinical samples

[0128] Conventional PCR and isothermal amplification detection based on the kit of Example 1 were performed on 20 samples of pigs with suspected symptoms of pseudorabies virus (PrV) and classical swine fever virus (CSFV) sent for clinical examination, and the test results are shown in Table 5. The results show that the isothermal amplification detection based on the embodiment of the present invention is more sensitive than conventional PCR, and can quickly perform detection and disease typing, and has high detection efficiency, which is of great significance for rapid isolation and targeted treatment.

[0129] Table 5 Comparison results of two detection methods

[0130]

[0131] All the operations on the above-mentioned pathological samples were completed at the China Animal Health and Epidemiology Center.

[0132] Embodiment 6:

[0133] like Fig.12 and Fig.14 As shown, the difference between this embodiment and the microfluidic chip in embodiment 1 is that the microfluidic chip in this embodiment is provided with a normally open stop valve 500 at the upstream section of the amplification flow channel 308. Fig.12 , Fig.15As shown, the stop valve 500 includes a valve seat 501, a valve core 502 and a valve membrane 503. The valve seat 501 is connected to the amplification flow channel 308. The valve core 502 can produce micro displacement in the up and down directions relative to the valve seat 501. The valve seat 501 is divided into 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 fixed to the diaphragm base 5012 by bonding. The valve membrane 503 corresponds to the valve seat body unit 5011 one by one. Fig.13 As shown, 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 respectively, and the lower end of the valve core 502 is a dome.

[0134] The diaphragm base 5012 is provided with a valve diaphragm positioning groove, and the valve diaphragm 503 is a circular piece. The valve diaphragm 503 includes a middle portion that can cover the connection pool 316, a fixed portion connected to the diaphragm base 5012, and a joint portion connecting the middle portion and the fixed portion, and the joint portion is in contact with the side wall 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 the bottom of the diaphragm base 5012 can be bonded to the top plate 200 by acrylic structural adhesive.

[0135] A valve cap 504 is provided at the upper end of the valve core 502, an external thread is provided at the upper part of the valve core 502, and an internal thread is provided at the valve seat body unit 5011. The valve cap 504 is screwed to make the lower end of the valve core 502 contact the middle part of the valve membrane 503 and push the middle part toward the connection pool 316. The diameter of the lower circular cross section of the valve core 502 is smaller than the diameter of the connection pool 316, and the lower peripheral side of the valve core 502 squeezes the joint part of the valve membrane 503 and is in close contact with the wall of the connection pool 316. The valve membrane 503 and the valve core 502 are separately provided. When the stop valve 500 is not closed, the stop valve 500 can provide a double seal to improve the sealing effect. A break line can be considered to be provided at the intersection of the fixed part and the joint part. The middle part of the valve membrane 503 is subjected to a downward force, and the joint part is separated from the fixed part at the break line, which can ensure the integrity of the middle part and the joint part of the valve membrane 503, and the sealing effect of the valve core 502 and the connection pool 316 is more reliable. When the valve membrane 503 is provided with an easy-to-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 an easy-to-break line, the valve membrane 503 only elastically deforms and remains intact after the stop valve 500 is closed, and the stop valve 500 can be reopened.

[0136] In this embodiment, there are two groups of stop valves 500, and each group of stop valves 500 corresponds to the amplification chamber group on that side. Fig.14As shown, the valve seat body units 501 in each stop valve 500 are arranged in four. To prevent the stop valve 500 from being started by mistake, a safety buckle 505 is arranged between the upper end of the valve seat body unit 5011 and the valve cap 504, as shown in FIG. Fig.16 As shown, the safety buckle 505 is U-shaped, and the height of the safety buckle 505 is adapted to the safety 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 part of the valve membrane 503. The U-shaped opening end of the safety buckle 505 has a C-shaped portion that shrinks inwardly, which can prevent the safety buckle 505 from accidentally falling off.

[0137] According to the preset time, after the sample (nucleic acid template) enters the amplification flow channel 308, the stop valve 500 is normally open, the safety buckle 505 is removed, and the valve cap 504 is manually screwed one by one to make the corresponding valve core 502 move downward, and the amplification flow channel 308 is closed through the valve core 502 and the valve membrane 503. Of course, when special equipment is not used, a mechanical thrust can be directly applied to the stop valve 500 manually with the help of special tools. The stop valve 500 can completely cut off the fluid channel after closing. The function of the stop valve 500 is to protect the equipment when a power failure occurs, and at the same time prevent the liquid in the amplification chamber 309 from flowing back due to other unexpected factors and causing interference.

[0138] Embodiment 7:

[0139] The difference between this embodiment and the microfluidic chip in embodiment 6 is that Fig.17 As shown, the upper part of the valve core 502 of the stop valve 500 in this embodiment is not connected to the valve seat body unit 5011 by threaded connection, but by a press-on connection. Pressing to close the stop valve 500 is easier and faster. The upper part 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 arranged around the lower part of the valve core 502. The flaps 506 take the lower part of the valve core 502 as the parent body, and the upper end of the flaps 506 is away from the lower part of the valve core 502 as the parent body and is not connected to the parent body. The end of the flaps 506 is integrated with the lower part 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 along the valve seat body unit 5011 by a small distance. 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 is reopened. 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 the 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.

[0140] After the sample (nucleic acid template) enters the amplification flow channel 308 at a predetermined time, the safety buckle 505 is removed, and the valve caps 504 are manually pressed one by one to make the corresponding valve cores 502 move downward, and the amplification flow channel 308 is closed through the valve cores 502 and the valve membrane 503. Alternatively, a dedicated trigger device is provided at the upper end of the corresponding valve caps 504 on the detection device, and a pressing force is uniformly applied to each valve cap 504 to realize the automatic closing of the stop valve 500.

[0141] Embodiment 8:

[0142] like Fig.18 and Fig. 20 As shown, the difference between this embodiment and the microfluidic chip in embodiment 1 is that the microfluidic chip in this embodiment is provided with a normally closed shutoff valve 600 at the downstream section of the extraction channel 306. The shutoff valve 600 is similar in structure to the stop valve 500 in embodiment 6, but the lower end of the shutoff valve core 602 of the shutoff valve 600 in this embodiment is covered by a shutoff valve membrane 603, and the shutoff valve membrane 603 can move together with the lower end of the shutoff valve core 602.

[0143] like Fig.19 As shown, the bottom of the shutoff valve seat 601 of the shutoff valve 600 of this embodiment is fixed on the top plate 200 and can be bonded by acrylic structural adhesive. The shutoff valve connecting hole 208 is set on the top plate 200, and the interception pool 317 is correspondingly set downstream of the extraction channel 306 of the fluid plate 300. The shutoff valve cap 604 is set at the upper end of the shutoff valve core 602, the upper part of the shutoff valve core 602 is set with an external thread, and the shutoff valve seat 602 is set with an internal thread. The shutoff valve cap 604 is screwed to make the lower end of the shutoff valve core 602 drive the shutoff valve membrane 603 to approach the interception pool 317. The diameter of the lower circular cross-section of the shutoff valve core 602 is smaller than the diameter of the interception pool 317, and the lower peripheral side surface and lower end surface of the shutoff valve core 602 squeeze the shutoff valve membrane 603 and the wall of the interception pool 317 in close contact.

[0144] The shut-off valve 600 is kept 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 flow chamber 307, the amplification flow channel 308 and the amplification chamber 309 is lower than the external atmospheric pressure. In this way, no external power source is required during use. Figure 5 and Figure 6Since the microfluidic matrix on the chip has been processed, the sample flow channel 302 and the buffer flow channel 304 located upstream of the extraction flow channel 306 generate adsorption force on the microfluid, and the vent 310 and the overflow hole 3054 ventilate the pressure in the fluid plate 300 to the 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 shut-off valve 600 is opened when the sample is added. The reagent powder in the extraction chamber 305 is fully re-dissolved, and the pressure of the shunt chamber 307 and the amplification flow channel 308 is low. The existence of the pressure difference will guide the liquid through the serpentine extraction flow channel 306, and efficiently mix to obtain a nucleic acid template for pathogen detection. The sample (nucleic acid template) flows into the two shunt chambers 307, and then through the amplification flow channel 308, enters the amplification chamber 309, and the constant temperature amplification reagent powder is dissolved. 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 isothermal amplification, comprising a microfluidic chip, characterized in that: The microfluidic chip is provided with a sample injection chamber, two buffer chambers, two extraction chambers, two shunt chambers, an amplification chamber, and a microfluidic flow channel that realizes sequential communication among the sample injection chamber, the buffer chamber, the extraction chamber, the shunt chamber, and the amplification chamber; the amplification chamber comprises at least six chambers, each of which is independent of each other, and each amplification chamber is pre-installed with a corresponding constant temperature amplification reagent powder, the constant temperature amplification reagent powder pre-installed in the amplification chamber can respectively amplify any one of the following six pathogens, and at the same time, the pathogens amplified by the constant temperature amplification reagent powder in all amplification chambers can The microfluidic chip 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; the isothermal amplification reagent powder includes primers, probes, DNA helicase and DNA polymerase for isothermal amplification of the above six pathogens; the outermost layer of the microfluidic chip is a shell, and the shell is composed of a top plate, a fluid plate and a bottom plate from top to bottom; the microfluidic flow channel includes a sample flow channel, a buffer flow channel, an extraction flow channel and an amplification flow channel, and the middle position on one side of the fluid plate is The sample injection chamber is provided, the two buffer chambers are connected to the sample injection chamber through the sample splitting flow channel, the two extraction chambers are connected to the corresponding buffer chambers through the buffer flow channel, the two split flow chambers are connected to the corresponding extraction chambers through the extraction flow channel, and the top plate and the shell are respectively provided with sample injection ports corresponding to the sample injection chamber; the amplification chamber is set into two groups, which are respectively located at the two ends of the other side of the fluid plate, and each group of amplification chambers is arranged in sequence along the end of the parallel fluid plate, and the amplification flow channel is connected to the amplification chamber one by one, and each amplification chamber is connected to the corresponding split flow channel through the amplification flow channel. The flow chamber is connected; the extraction chamber includes an active mixing zone, a dry powder pre-setting zone and an overflow zone, the active mixing zone is provided with a mixing step, 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, the extraction flow channel is a serpentine flow channel, the buffer flow channel is connected with the active mixing zone, the extraction flow channel is connected with the dry powder pre-setting zone, and the dry powder pre-setting zone is pre-set with corresponding nucleic acid release reagent dry powder.

2. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 1, characterized in that: The bottom plate is provided with a waste liquid chamber and a clearance groove, the waste liquid chamber is provided with an absorption pad, the overflow hole is communicated with the waste liquid chamber; the clearance groove is located below the amplification chamber.

3. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 2, characterized in that: 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 arranged at the corresponding positions of the ventilation holes on the shell and the top plate.

4. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 3, characterized in that: The top plate is also provided with an air flow channel communicating with the flow distribution 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.

5. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 4, characterized in that: 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 are provided with openings for the end of the fluid infusion tube to pass through, the end of the fluid infusion tube is sealed with a dissolving film, the top end of the fluid infusion tube is provided with a sealing cover, the upper section of the fluid infusion tube is a corrugated tube, and the lower section is conical, the end of the fluid infusion tube faces the fluid infusion chamber of the fluid plate, a needle-shaped object is provided in the fluid infusion chamber of the fluid plate at the center of the end of the fluid infusion tube, and the bypass flow channel connects the fluid infusion chamber and the two extraction flow channels.

6. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to any one of claims 1 to 5, characterized in that: The fluid plate is also provided with a heat isolation area, which includes a plurality of heat isolation grooves, and the plurality of heat isolation grooves surround the amplification flow channel and the amplification chamber respectively.

7. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 6, characterized in that: The shell surface is provided with a label covering area, to which a label is attached; the shell is provided with a sample indicating area, which is located beside the sample injection chamber; and the shell is provided with a detection port.

8. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 7, characterized in that: A normally open stop valve is arranged 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 a plurality of valve seat body units connected together, the diaphragm base is fixed on the top plate, the number of the 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, the valve membrane corresponds to the valve seat body unit one by one, and when the stop valve is closed, the valve core and the valve membrane cut off the amplification flow channel.

9. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 7 or 8, characterized in that: A normally closed shut-off valve is arranged on the extraction flow channel, and the shut-off valve comprises 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 is integrally wrapped with the lower end of the shut-off valve core. When the shut-off valve is opened, the extraction flow channel is communicated with the diversion chamber.

10. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 1, 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; 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.

11. The microfluidic chip kit for rapid nucleic acid detection based on recombinase isothermal amplification according to claim 10, characterized in that: Two of the amplification chambers on the microfluidic chip are configured as a positive control chamber and a negative control chamber, wherein the positive control chamber is pre-installed with a positive quality control product containing plasmids corresponding to the amplified gene sequences of the above six pathogens.

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