Microfluidic chip and influenza a virus detection kit

Abstract

The present application belongs to the field of in vitro diagnostics, and particularly relates to a microfluidic chip and an influenza A virus detection kit. The chip comprises a cover plate, a first holding plate, a second holding plate and a bottom plate arranged in sequence from top to bottom, wherein the cover plate is provided with a first sample-loading cavity; the first holding plate is provided with a second sample-loading cavity, a first buffer cavity and a test paper cavity that are connected in sequence by means of a flow channel; and the second holding plate is provided with a reaction cavity, a second buffer cavity and a water absorption cavity that are connected in sequence by means of a flow channel. The microfluidic chip is ingeniously configured as an integrated small detection device. The structure of the microfluidic chip is located on different chip layers, thereby ensuring the independent operation of each operation unit. The aligned buffer cavities are provided between two interlayers, and the design of the buffer cavities enables a product to be fully and uniformly diluted; a sliding device can precisely control the release of a dilution; and the water absorption cavity can absorb the redundant dilution, thereby ensuring that a chromatography test strip can clearly and accurately display the result. By means of the device, the effects of simplifying test steps and preventing contamination can be achieved.

Description

Microfluidic chips and influenza A virus detection kits Technical Field

[0001] This application belongs to the field of in vitro diagnostics, specifically relating to a microfluidic chip and an influenza A virus detection kit. Background Technology

[0002] Influenza A virus (H1N1) is a common influenza virus with RNA as its genetic material. It includes multiple subtypes and can infect various mammals, including wild birds, domestic poultry, pigs, horses, and humans, causing influenza. H1 and H3 influenza viruses are among the known H1N1 viruses capable of transmission between humans. Human infection with influenza viruses causes influenza, leading to respiratory illnesses that can be life-threatening in severe cases. Influenza outbreaks are seasonal and spread rapidly, making rapid diagnosis essential for timely treatment and interrupting the spread of the disease.

[0003] Molecular diagnostics primarily uses molecular biology methods to detect the presence of genetic material, including changes in its structure and expression levels. Its advantages lie in its excellent sensitivity and accuracy, and it is mainly used for the detection and diagnosis of infectious diseases, genetic diseases, and tumors. Currently, the main methods for molecular diagnostics include real-time PCR, isothermal amplification, fluorescence in situ hybridization, gene chips, and sequencing technology.

[0004] The gold standard for nucleic acid detection in existing technologies is quantitative real-time PCR (qPCR), which is widely used for the detection of various pathogens due to its excellent sensitivity and accuracy. However, the application of qPCR has the following limitations: (1) it requires expensive instruments; (2) it requires professional personnel to perform the experiments; (3) it has high requirements for the experimental environment and requires clear experimental zoning; and (4) it takes a long time from sampling to result output, which cannot meet the needs of real-time detection. These conditions limit the flexible application of this technology in other scenarios, such as primary healthcare institutions, medical facilities in remote areas, and institutions that need to obtain test results quickly.

[0005] Other methods, such as colloidal gold immunochromatography for antigen-antibody detection, are simple to operate and can quickly obtain test results, but their sensitivity and specificity are relatively poor.

[0006] Therefore, in order to solve the above problems, there is an urgent need for a simple, rapid, sensitive and accurate detection method to achieve rapid diagnosis of pathogens in different situations.

[0007] Summary of the Invention

[0008] Based on this, one embodiment of this application provides a microfluidic chip and an influenza A virus detection kit.

[0009] The microfluidic chip comprises, from top to bottom, a cover plate, a first clamping plate, a second clamping plate and a bottom plate.

[0010] The cover plate is provided with a first sample loading cavity.

[0011] The first clamping plate is provided with a second sample loading cavity, a first buffer cavity and a test paper cavity connected in sequence by flow channels.

[0012] The second clamping plate is provided with a reaction cavity, a second buffer cavity and a water absorption cavity connected in sequence by flow channels.

[0013] The first sample loading cavity, the second sample loading cavity and the reaction cavity are in communication; the first buffer cavity and the second buffer cavity are in communication; and the test paper cavity and the water absorption cavity are in communication.

[0014] In one embodiment, the test paper cavity is provided with an immunochromatographic test paper.

[0015] In one embodiment, the second clamping plate is further provided with a sliding cavity connected to the reaction cavity by a flow channel, and the sliding cavity is provided with a sliding device and a diluent package; the sliding device is in dynamic sealing with the sliding cavity, and the sliding device can move under the action of an external force to pierce the diluent package, so that the diluent flows from the sliding cavity into the reaction cavity.

[0016] In one embodiment, the sliding device is provided with a piercing member, and when a pressure is applied to the diluent package towards the piercing member, the piercing member can pierce the diluent package, and the diluent in the diluent package can flow into the reaction cavity, the second buffer cavity and the first buffer cavity.

[0017] In one embodiment, the cover plate is further provided with an air outlet hole in communication with the reaction cavity, for discharging air inside the chip and maintaining pressure.

[0018] The application further provides an application of the above-mentioned microfluidic chip in pathogen detection.

[0019] The application further provides a pathogen detection kit comprising the above-mentioned microfluidic chip.

[0020] The application further provides an influenza A virus detection kit comprising the above-mentioned microfluidic chip and an influenza A virus nucleic acid detection reagent.

[0021] In one embodiment, the influenza A nucleic acid detection reagent comprises a detection primer pair, and the detection primer pair comprises:

[0022] an outer primer 1, a nucleotide sequence of the outer primer 1 is shown as SEQ ID NO. 1;

[0023] an outer primer 2, a nucleotide sequence of the outer primer 2 is shown as SEQ ID NO. 2;

[0024] an inner primer 3, a nucleotide sequence of the inner primer 3 is shown as SEQ ID NO. 3;

[0025] an inner primer 4, a nucleotide sequence of the inner primer 4 is shown as SEQ ID NO. 4;

[0026] a loop primer 5, a nucleotide sequence of the loop primer 5 is shown as SEQ ID NO. 5;

[0027] a loop primer 6, a nucleotide sequence of the loop primer 6 is shown as SEQ ID NO. 6.

[0028] a 5' end of the inner primer 3 or the inner primer 4 is labeled with a chemical modification group, and a 5' end of the loop primer 5 or the loop primer 6 is labeled with a fluorescent group.

[0029] Optionally, a 5' end of the inner primer 3 or the inner primer 4 is labeled with a fluorescent group, and a 5' end of the loop primer 5 or the loop primer 6 is labeled with a chemical modification group.

[0030] In one of the embodiments, the chemical modification group comprises one of biotin and digoxin; and the fluorescent group comprises one of FAM and FITC.

[0031] In one of the embodiments, the kit further comprises a buffer solution.

[0032] The buffer solution comprises 8mM-12mM of (NH4)2SO4, 40mM-60mM of KCl, 5mM-12mM of MgSO4, 0.05v / v%-0.15v / v% of Tween-20, 0.5mg / mL-1.5mg / mL of BSA and 50mM-150mM of L-Proline.

[0033] The application provides a microfluidic chip, which comprises a cover plate, a first clamping plate, a second clamping plate and a bottom plate from top to bottom, and is a one-piece small detection device. The structure is located in different chip layers, which ensures that each operation unit works independently and reduces interference. There is an aligned buffer cavity between the two clamping layers. The design of the buffer cavity allows the product to be fully and uniformly diluted. The sliding device can accurately control the release of the diluent, and the water absorption chamber can absorb the excess diluent, ensuring that the chromatography test strip result is clear and accurate. By using the device, the experimental steps can be simplified, and the pollution of aerosol generated during the product transfer process can be avoided.

[0034] The device is simple to operate, and the result reading is direct and does not depend on complex and expensive experimental equipment, so it is suitable for flexible application in various detection scenes and provides a new detection device for POCT application. Moreover, the reaction process and result reading steps can be combined to simplify the experimental steps. Further, the microfluidic chip designed in the application combined with the influenza A virus detection reagent can realize rapid nucleic acid detection, prevent aerosol pollution in the nucleic acid amplification and transfer process, and has high sensitivity and specificity. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0036] Fig. 1 is a schematic diagram of the microfluidic chip as a whole;

[0037] Fig. 2 is a disassembled view of each part of the microfluidic chip;

[0038] Fig. 3 is a schematic diagram of the amplification results of the influenza A virus positive sample (a) and negative sample (b) using the system designed in the present application;

[0039] Fig. 4 is a test result diagram of the influenza A virus positive sample (a) and negative sample (b) using the microfluidic chip;

[0040] Fig. 5 is a sensitivity test result diagram;

[0041] Fig. 6 is a fluorescence method sensitivity test result diagram;

[0042] Fig. 7 is a specificity verification result diagram;

[0043] Fig. 8 is a detection result diagram of RNA samples (1, 2, 3, 4) and DNA plasmid samples containing target fragments (1, 2, 3), respectively;

[0044] Legend: microfluidic chip 10 cover plate 11; first sample cavity 111; air outlet hole 112; first clamp plate 12; sliding cavity 121; second sample cavity 122; first buffer cavity 123; test paper cavity 124; sliding device 125; piercing member 126; displacement slot 1251; 13, second clamp plate; reaction cavity 131; second buffer cavity 132; water absorption cavity 133; 14, bottom plate. DETAILED DESCRIPTION

[0045] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the application and not intended to limit the scope of the application. The purpose of providing these embodiments and examples is to make the disclosure of the application more thoroughly and comprehensively understood. It should also be understood that the application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the application, and the equivalent forms obtained thereby also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application. It should be understood that the application can be implemented without one or more of these details.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0047] The term

[0048] All documents referred to in this application are incorporated by reference in this application as if each document were individually incorporated by reference. Unless otherwise stated, the documents referred to in this application are incorporated by reference in their entirety, for all purposes. When the documents referred to in this application are referred to, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the documents referred to in this application are referred to, the examples and preferred modes of the relevant technical features that are referred to can also be incorporated by reference into this application, but are limited to the implementation of this application. It should be understood that when the content referred to conflicts with the description in this application, the application is given priority or is modified according to the description in this application.

[0049] Unless otherwise stated or contradictory, the terms or phrases used herein have the following meanings:

[0050] The selection scope of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0051] In the present application, when referring to a numerical interval (i.e. a numerical range), if no specific description is provided, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical end points. If no specific description is provided, when the numerical interval only refers to the integers within the numerical interval, including the two end point integers of the numerical range and each integer between the two end points, in the present application, each integer is directly listed, for example, t is an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges included therein.

[0052] The term "loop-mediated isothermal amplification (LAMP)" is a new nucleic acid amplification technology, which has been widely used in the fields of pathogenic microorganism detection and infectious disease diagnosis. Its characteristics are that four or six primers are designed for six regions of the target sequence, and high-efficiency amplification of the target sequence can be realized under constant temperature conditions by relying on Bst DNA polymerase with high strand displacement activity. LAMP technology is very suitable for rapid diagnosis in flexible scenarios because it does not depend on complex temperature cycling instruments and simplifies the requirements for experimental conditions.

[0053] The term "microfluidic" microfluidic technology is a new technology that can accurately control and manipulate microscale fluid. The preparation, separation, reaction and detection of samples are realized in a micro space, and multiple operation units are integrated in a chip. Microfluidic technology involves the intersection of chemistry, biology, fluid physics, microelectronics, new materials science and biomedical engineering.

[0054] Microfluidic technology has now been applied in the field of in vitro diagnosis, and the nucleic acid detection system based on microfluidic chip is one of the most widely used applications. The technology platform integrating microfluidic chip and LAMP technology has the advantages of strong specificity, high sensitivity, short time, simple operation, etc., and has great application prospect in point of care testing (POCT).

[0055] The term "immunochromatography" is a simple and rapid diagnostic method, and is also an important tool for point of care testing (POCT). It was first used for the detection of human chorionic gonadotropin (HCG) as an early diagnosis of pregnancy.

[0056] Immunochromatography technology is mainly completed by immunochromatography test strips. Immunochromatography technology is not only simple, rapid, specific, stable, but also convenient to carry, and has been widely used in clinical diagnosis, food safety, drug detection, environmental pollution and other fields.

[0057] One aspect of the present application provides a microfluidic chip, which comprises, from top to bottom, a cover plate, a first clamping plate, a second clamping plate and a bottom plate; the cover plate is provided with a first sample loading cavity; the first clamping plate is provided with a second sample loading cavity, a first buffer cavity and a test paper cavity connected in sequence by flow channels; the second clamping plate is provided with a reaction cavity, a second buffer cavity and a water absorption cavity connected in sequence by flow channels.

[0058] The first sample loading cavity, the second sample loading cavity and the reaction cavity are all communicated; the first buffer cavity and the second buffer cavity are communicated; the test paper cavity and the water absorption cavity are communicated, so as to synthesize four layers of structure into an integrated microfluidic detection chip. And along the fluid advancing direction, the liquid in the upstream cavity enters the downstream cavity by applying pressure to the microfluidic chip.

[0059] In a specific example, the test paper cavity is provided with an immunochromatography test paper. For example, a colloidal gold conjugated anti-FITC IgG antibody, a T line is streptavidin, and a C line is a goat anti-rabbit IgG antibody.

[0060] The second clamping plate further has a sliding cavity, the sliding cavity is connected with the reaction cavity through a flow channel, the sliding cavity is provided with a sliding device and a dilution liquid packaging bag, the sliding device realizes dynamic sealing with the sliding cavity, and the sliding device can move under the action of external force to pierce the dilution liquid packaging bag, so that the dilution liquid enters the reaction cavity from the sliding cavity.

[0061] Optionally, the cover plate is further provided with an air outlet hole. The air outlet hole is communicated with the reaction cavity, and is used for discharging the gas in the chip to maintain the pressure.

[0062] In a specific example, the sliding cavity is provided with a dilution liquid packaging bag. For example, a liquid capsule packaged by an aluminum foil bag, which is provided with the dilution liquid.

[0063] In a specific example, the sliding device is provided with a piercing member. When the pressure towards the piercing member is applied to the dilution liquid packaging bag, the piercing member can pierce the dilution liquid packaging bag, and the dilution liquid in the dilution liquid packaging bag can flow into the reaction cavity, the second buffer cavity and the first buffer cavity.

[0064] The sliding device is provided with a displacement slot, and correspondingly, the second clamping plate 13, the bottom plate 14 and the first clamping plate 12 are provided with notches. In use, the operator can conveniently push the sliding device through the displacement slot to apply the pressure towards the piercing member more labor-savingly. Correspondingly, the notches can make it more convenient to push the displacement slot to the accurate position, and when the displacement slot collides with the related notch, the release of the dilution liquid packaging bag is realized.

[0065] Optionally, the sliding device is an embedded plug.

[0066] The structure of the microfluidic chip in the application is located in different chip layers, so that the operation units work independently and the interference is reduced. The aligned buffer chambers are arranged between the two clamping layers, and the design of the buffer chambers enables the product to be fully and uniformly diluted. The chip further comprises a sliding device (embedded plug), and the end of the plug is provided with a piercing member (piercing needle) for releasing the dilution liquid in the dilution liquid packaging bag. The sliding device (embedded plug) is pushed into the microfluidic chip, so that the release of the dilution liquid can be accurately controlled. The water absorption pad chamber can absorb the excess dilution liquid, so as to ensure that the result of the chromatographic test strip is clearly and accurately displayed. By using the device, the experimental steps can be simplified, and the pollution such as aerosol generated in the product transfer process can be avoided.

[0067] The application further provides a pathogen detection kit comprising the microfluidic chip.

[0068] Another aspect of the application further provides an influenza A virus detection kit comprising the microfluidic chip and an influenza A virus nucleic acid detection reagent.

[0069] The application analyzes the matrix gene of influenza A virus, selects a specific and conserved segment with no similarity to other viruses as a target region. The primers based on loop-mediated isothermal amplification (LAMP) are designed, and six primers are designed for six target regions, which can specifically bind to the genome of influenza A virus and produce amplification products.

[0070] The nucleotide sequence of the target sequence is shown in SEQ ID NO. 7:

[0071] Alternatively, the influenza A virus nucleic acid detection reagent comprises a detection primer pair, which comprises:

[0072] an outer primer 1, the nucleotide sequence of which is shown in SEQ ID NO. 1;

[0073] an outer primer 2, the nucleotide sequence of which is shown in SEQ ID NO. 2;

[0074] an inner primer 3, the nucleotide sequence of which is shown in SEQ ID NO. 3;

[0075] an inner primer 4, the nucleotide sequence of which is shown in SEQ ID NO. 4;

[0076] a loop primer 5, the nucleotide sequence of which is shown in SEQ ID NO. 5;

[0077] a loop primer 6, the nucleotide sequence of which is shown in SEQ ID NO. 6;

[0078] In one specific example, the 5' end of the inner primer 3 or the inner primer 4 is labeled with a chemical modification group, and the 5' end of the loop primer 5 or the loop primer 6 is labeled with a fluorescent group;

[0079] Alternatively, the 5' end of the inner primer 3 or the inner primer 4 is labeled with a fluorescent group, and the 5' end of the loop primer 5 or the loop primer 6 is labeled with a chemical modification group.

[0080] Alternatively, the chemical modification group includes but is not limited to one of biotin and digoxin; and the fluorescent group includes one of FAM and FITC.

[0081] In one specific example, it further comprises a buffer;

[0082] The buffer includes 8mM-12mM of (NH4)2SO4, 40mM-60mM of KCl, 5mM-12mM of MgSO4, 0.05v / v%-0.15v / v% of Tween-20, 0.5mg / mL-1.5mg / mL of BSA and 50mM-150mM of L-Proline.

[0083] The specific primers are shown in Table 1:

[0084] Table 1

[0085] For example, 8mM, 9mM, 10mM, 11mM, 12mM of (NH4)2SO4;

[0086] 40mM, 45mM, 50mM, 55mM, 60mM of KCl;

[0087] 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM of MgSO4;

[0088] 0.05v / v%, 0.06v / v%, 0.07v / v%, 0.08v / v%, 0.09v / v%, 0.10v / v%, 0.11v / v%, 0.12v / v%, 0.13v / v%, 0.14v / v%, 0.15v / v% of Tween-20;

[0089] 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL, 1.0mg / mL, 1.1mg / mL, 1.2mg / mL, 1.3mg / mL, 1.4mg / mL, 1.5mg / mL of BSA;

[0090] 50mM, 55mM, 60mM, 65mM, 70mM, 85mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM of L-Proline.

[0091] The detection reagent system is configured:

[0092] Including buffer ((NH4)2SO4, KCl, MgSO4, Tween-20, BSA, L-Proline), dNTP, outer primer 1, outer primer 2, inner primer 3, inner primer 4, loop primer 5, loop primer 6, Bst DNA polymerase, reverse transcriptase, RNase inhibitor.

[0093] Reaction condition: 60-65℃ for 30 minutes.

[0094] Detection of product by chromatographic test strip

[0095] Chromatographic test strip condition: colloidal gold conjugated anti-FITC IgG antibody, T line is streptavidin, C line is goat anti-rabbit IgG antibody.

[0096] After diluting the amplification product 10-20 times with PBS (pH 7.4), 50 μL of the diluted solution was dropped onto the sample pad of the chromatographic test strip, and the sample was allowed to chromatograph, and the results were detected after 5 minutes.

[0097] Positive result: both T line (Test line) and C line (Control line) show bands.

[0098] Negative result: only C line (Control line) shows bands.

[0099] II. Microfluidic integrated device

[0100] The microfluidic device comprises four layers of chips: the overall schematic diagram of the device is shown in Figure 1. The specific structure is shown in Figure 2, which comprises, from top to bottom, a cover plate, a first clamping plate, a second clamping plate and a bottom plate;

[0101] The cover plate is provided with a first sample loading cavity;

[0102] The first clamping plate is provided with, in sequence through flow channels, a sliding cavity, a second sample loading cavity, a first buffer cavity and a test paper cavity;

[0103] The second clamping plate is provided with, in sequence through flow channels, a reaction cavity, a second buffer cavity and a water absorption cavity;

[0104] Among them, the first sample loading cavity is communicated with the second sample loading cavity; the first buffer cavity is communicated with the second buffer cavity; the first sample loading cavity and the second sample loading cavity are both communicated with the reaction cavity, and the test paper cavity is communicated with the water absorption cavity.

[0105] Working steps of the microfluidic device:

[0106] (1) Cover the sample loading hole with a sticker, lift the sticker, and add 20 μL of the mixed sample and reaction liquid mixture through the sample loading hole, and the mixed liquid reaches the reaction cavity of the second clamping plate through the sample loading hole.

[0107] (2) Place the microfluidic device in a reaction instrument or a 60-65℃ environment for reaction for 30 minutes.

[0108] (3) The first clamping plate has a push piece, and the push piece has an aluminum foil bag encapsulated liquid capsule in front of the push piece, wherein the liquid capsule contains 200 μL of diluent. The push piece is pushed to the innermost side, the needle pierces the liquid capsule to release the diluent, and the diluent is pushed into the lower second clamping plate reaction chamber, and then into the buffer chamber. The second clamping plate buffer chamber is in communication with the first clamping plate buffer chamber, and the diluent then passes through the first clamping plate buffer chamber into the test strip chamber for chromatography.

[0109] (4) Excess sample diluent will be absorbed by the absorbent pad embedded in the absorbent pad chamber.

[0110] (5) The chromatographic test strip result can be read after 5 minutes.

[0111] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the examples are only used to illustrate but not to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0112] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed for the temperature and time parameters.

[0113] It should be understood that the order of the above processes in various embodiments of the present application does not mean the execution sequence, and the execution sequence of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0114] Example 1

[0115] A microfluidic chip comprises, from top to bottom, a cover plate 11, a first clamping plate 12, a second clamping plate 13, and a bottom plate 14.

[0116] The cover plate 11 is provided with a first sample loading chamber 111.

[0117] The first clamping plate 12 is provided with, in sequence from top to bottom, a sliding chamber 121, a second sample loading chamber 122, a first buffer chamber 123, and a test strip chamber 124 through flow channels.

[0118] The second clamping plate 13 is provided with, in sequence from top to bottom, a reaction chamber 131, a second buffer chamber 132, and an absorbent chamber 133 through flow channels.

[0119] The first sample loading cavity 111 is communicated with the second sample loading cavity 122. The first buffer cavity 123 is communicated with the second buffer cavity 132. The first sample loading cavity 111 and the second sample loading cavity 122 are both communicated with the reaction cavity 131, and the test paper cavity 124 is communicated with the water absorption cavity 133.

[0120] The test paper cavity 124 is provided with an immunochromatographic test paper.

[0121] The sliding cavity 121 is provided with a sliding device 125 and a diluent packaging bag.

[0122] The sliding device is provided with a slot 1251. Correspondingly, the second clamping plate 13, the bottom plate 14 and the upper surface of the first clamping plate 12 are provided with notches. The sliding device 125 is provided with a piercing member 126. When a pressure is applied to the diluent packaging bag towards the piercing member 126, the piercing member 126 can pierce the diluent packaging bag. The diluent in the diluent packaging bag can flow into the reaction cavity 131, and then pass through the second buffer cavity 132 and the first buffer cavity 123, and finally enter the test paper cavity 124.

[0123] The cover plate is further provided with an air outlet hole 112.

[0124] II. Detection process

[0125] The extraction of the virus genome is performed according to the operation steps of the virus extraction kit. The obtained nucleic acid is used as a sample for the next reaction.

[0126] The positive sample (a) and the negative sample (b) of the influenza A virus are amplified by using the system designed in the present application. After the amplification product is diluted by 10 times, 50 μL of the diluted product is taken for the chromatographic test paper strip detection. The positive (+) and negative (-) results can be clearly displayed, and the interpretation method is simple.

[0127] The reaction system 20 μL: the reaction system includes buffer (NH4)2SO4(10 mM), KCl (50 mM), Tween-20 (0.1% v / v), BSA (1 mg / mL), L-Proline (100 mM), MgSO4(8 mM); dNTP (1.4 mM), primers 1, 2 (0.2 μM), primers 3, 4 (1.6 μM), primers 5, 6 (0.8 μM), Bst enzyme (6 U / reaction), reverse transcriptase (7.5 U / reaction), RNase inhibitor (10 U / reaction), sample 10 μL, and deionized water is added to make up to 20 μL.

[0128] The reaction program is 65℃ for 30 min. The experimental results are shown in FIG. 3.

[0129] The results show that the positive sample amplification product dilution chromatographic test strip T line appears a band (+), while the negative sample does not produce a corresponding band (-). It shows that the influenza A virus detection kit has the ability to detect influenza A virus.

[0130] Result interpretation: If the test result shows that a band appears at the T line position, the sample can be judged as a virus positive sample; if no band appears at the T line position, the sample can be judged as a non-detectable negative sample.

[0131] Similarly, using a microfluidic chip for testing, influenza A virus positive samples (a) and negative samples (b) are detected. The sample and reaction system are added to the reaction cavity, the chip is placed at 65 ° for reaction, and after the reaction is completed, the sliding chip is pushed in, and the dilution product is detected by chromatographic test strip. The detection results can clearly show the positive (+) and negative (-) results, and the interpretation method is simple, and the results are shown in Figure 4.

[0132] II. Sensitivity test

[0133] After the quantified influenza A virus samples are gradient diluted, the nucleic acid extraction is performed according to the operation guide of the virus nucleic acid extraction kit. The extracted nucleic acid is added to the prepared reaction system. The sample concentration is 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL and NTC (no template control).

[0134] Reaction system 20 μL: The reaction system includes buffer, Mg 2+ (8 mM), dNTP (1.4 mM), primers 1, 2 (0.2 μM), primers 3, 4 (1.6 μM), primers 5, 6 (0.8 μM), Bst enzyme (6 U / reaction), reverse transcriptase (7.5 U / reaction), sample 10 μL, and deionized water to 20 μL.

[0135] Reaction program: 65 °C for 30 minutes.

[0136] Experimental results: The product is diluted by ten times, and the test paper strip is used for result detection, and the results are shown in Figure 5. The sensitivity of the method is 10 3 copies / mL, which is consistent with the fluorescence detection result as shown in Figure 6.

[0137] III. Specificity detection

[0138] Specificity verification was carried out with influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, adenovirus nucleic acid and plasmid containing the target fragment. The primers involved in the application can only specifically amplify influenza A virus nucleic acid and positive control, and have no amplification signal for other respiratory viruses. It is shown that the primers have good specificity and no cross reaction with other respiratory viruses. The results are shown in Figure 7.

[0139] Comparison of sample testing and fluorescent method:

[0140] The influenza A virus detection reagent involved in the application was used to detect RNA samples (1, 2, 3, 4) and DNA plasmid samples containing the target fragment (1, 2, 3), and the results were compared with the LAMP constant temperature amplification fluorescence method. The results are shown in Figure 8 and Table 2. The results show that the results are consistent.

[0141] Table 2

[0142] The above-described embodiments only express several embodiments of the application, facilitate specific and detailed understanding of the technical solutions of the application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are within the scope of protection of the application. In addition, it should be understood that after reading the above teaching content of the application, those skilled in the art can make various changes or modifications to the application, and the equivalent forms obtained are also within the scope of protection of the application. It should also be understood that those skilled in the art can obtain technical solutions on the basis of the technical solutions provided by the application through logical analysis, reasoning or limited experiments, which are within the scope of protection of the appended claims of the application. Therefore, the scope of protection of the patent of the application should be based on the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A microfluidic chip, characterized by, The cover plate, the first clamping plate, the second clamping plate and the bottom plate are sequentially arranged from top to bottom. The cover plate is provided with a first sample loading cavity. The first clamping plate is provided with a second sample loading cavity, a first buffer cavity and a test paper cavity which are sequentially connected by flow channels. The second clamping plate is provided with a reaction cavity, a second buffer cavity and a water absorption cavity which are sequentially connected by flow channels. The first sample loading cavity, the second sample loading cavity and the reaction cavity are in communication; the first buffer cavity and the second buffer cavity are in communication; and the test paper cavity and the water absorption cavity are in communication.

2. The microfluidic chip of claim 1, wherein, The test paper cavity is provided with an immunochromatographic detection test paper.

3. The microfluidic chip of claim 1, wherein, The second clamping plate is further provided with a sliding cavity which is connected with the reaction cavity by a flow channel, and the sliding cavity is provided with a sliding device and a diluent package; the sliding device is in dynamic sealing with the sliding cavity, and the sliding device can move under the action of an external force to pierce the diluent package so that the diluent flows from the sliding cavity into the reaction cavity.

4. The microfluidic chip of claim 3, wherein, The sliding device is provided with a piercing member, and when a pressure is applied to the diluent package towards the piercing member, the piercing member can pierce the diluent package, and the diluent in the diluent package can flow into the reaction cavity, the second buffer cavity and the first buffer cavity.

5. The microfluidic chip according to any one of claims 1 to 4, wherein The cover plate is further provided with an air outlet hole which is in communication with the reaction cavity and is used for discharging air inside the chip to maintain the pressure.

6. Application of the microfluidic chip of any one of claims 1-5 in pathogen detection.

7. A pathogen detection kit, characterized in that, A kit comprising the microfluidic chip of any one of claims 1-5.

8. An influenza A virus detection kit, characterized by comprising: The kit comprises the microfluidic chip of any one of claims 1-5 and an influenza A virus nucleic acid detection reagent.

9. The influenza A virus detection kit according to claim 8, characterized in that, The influenza A virus nucleic acid detection reagent comprises a detection primer pair, and the detection primer pair comprises: an outer primer 1, the nucleotide sequence of which is shown in SEQ ID NO. 1; an outer primer 2, the nucleotide sequence of which is shown in SEQ ID NO. 2; an inner primer 3, the nucleotide sequence of which is shown in SEQ ID NO. 3; an inner primer 4, the nucleotide sequence of which is shown in SEQ ID NO. 4; a loop primer 5, the nucleotide sequence of which is shown in SEQ ID NO. 5; and a loop primer 6, the nucleotide sequence of which is shown in SEQ ID NO.

6.

10. The influenza A virus detection kit according to claim 8, characterized by The 5' end of the inner primer 3 or the inner primer 4 is labeled with a chemical modification group, and the 5' end of the loop primer 5 or the loop primer 6 is labeled with a fluorescent group. Alternatively, the 5' end of the inner primer 3 or the inner primer 4 is labeled with a fluorescent group, and the 5' end of the loop primer 5 or the loop primer 6 is labeled with a chemical modification group.

11. The influenza A virus detection kit according to claim 10, characterized in that, The chemical modification group comprises one of biotin and digoxin. The fluorescent group comprises one of FAM and FITC.

12. The influenza A virus detection kit according to any one of claims 8 to 11, characterized in that, Also included is a buffer; the buffer includes 8 mM to 12 mM of (NH4)2SO4, 40 mM to 60 mM of KCl, 5 mM to 12 mM of MgSO4, 0.05 v / v % to 0.15 v / v % of Tween-20, 0.5 mg / mL to 1.5 mg / mL of BSA, and 50 mM to 150 mM of L-Proline. Also included is a buffer; the buffer includes 8 mM to 12 mM of (NH4)2SO4, 40 mM to 60 mM of KCl, 5 mM to 12 mM of MgSO4, 0.05 v / v % to 0.15 v / v % of Tween-20, 0.5 mg / mL to 1.5 mg / mL of BSA, and 50 mM to 150 mM of L-Proline.

Images