WO2024066083A1

WO2024066083A1 - Constant-temperature detection kit and detection method for detecting pathogens

Info

Publication number: WO2024066083A1
Application number: PCT/CN2022/140205
Authority: WO
Inventors: 陈浩; 郭文浒; 刘华勇; 薛怡婷; 王红; 康洽惠; 林炳辉
Original assignee: 福州奥吉芯生物科技有限公司
Priority date: 2022-09-29
Filing date: 2022-12-20
Publication date: 2024-04-04

Abstract

The present invention relates to the technical field of biology, and in particular to a constant-temperature detection kit and detection method for detecting pathogens. The kit comprises an amplification system; the amplification system comprises a primer set; and the primer set is any one of an African swine fever virus primer set, a porcine circovirus primer set, a pseudorabies virus primer set or a porcine parvovirus primer set, and a SARS-CoV-2 primer set. According to the detection method, lyophilized beads of a virus amplification system are loaded in a micro-fluidic chip reaction tank, the lyophilized beads containing amplification detection components for viruses; and visual endpoint qualitative detection is performed on pathogens by means of H+ ions generated in an amplification process.

Description

Constant temperature detection kit and detection method for detecting pathogens

Technical Field

The invention relates to the field of biotechnology, and in particular to a constant temperature detection kit and a detection method for detecting pathogens.

Background technique

Pathogen detection can be divided into livestock and poultry pathogen detection, human pathogen detection, etc. Among them, the common livestock and poultry pathogens are swine pathogens, and the common human pathogens are new coronaviruses. Infection with the above pathogens will cause great harm to the social economy and human health.

African swine fever virus (ASFV), porcine circovirus (PCV), pseudorabies virus (PRV or ADV), and porcine parvovirus (PPV) are all common pathogens of pig infection. When detecting the above pig pathogens, most of them use PCR detection. For example, the Chinese invention patent with publication number CN106957927A discloses an African swine fever fluorescent PCR detection reagent, an African swine fever fluorescent PCR detection kit and its application, which can detect multiple samples at a time and is suitable for the detection of large quantities of samples. However, when using this method for detection, it is necessary to rely on a PCR instrument or an expensive real-time quantitative PCR instrument, and its various supporting equipment, a dedicated PCR laboratory and professional operators, and its cost and scope of application are subject to certain limitations. In addition, the detection cycle is long and it is not suitable for rapid on-site detection.

Nucleic acid is currently the main means of detecting the new coronavirus (SARS-COV-2) and is widely used in the screening of suspected patients, verification of cure, and environmental monitoring stations. The most commonly used nucleic acid detection method is the fluorescent quantitative probe method. As pointed out in the Chinese invention patent with announcement number CN112159868B, the fluorescent quantitative PCR instrument is used to collect the fluorescent signal generated by the cutting of nucleic acid probes during DNA synthesis in real time. It has the characteristics of high sensitivity and high specificity, but it has high requirements for equipment and is relatively complex to operate. This has caused great obstacles to the promotion and use of grassroots units. It is not suitable for rapid on-site detection, especially difficult to respond to public health emergencies.

In order to further improve the level of pathogen detection and achieve more convenient and rapid pathogen detection, various isothermal amplification technologies are increasingly attracting attention and their application scope is constantly expanding. On the one hand, isothermal amplification generally uses a constant temperature reaction mode to achieve amplification, which significantly reduces the complexity of the device; on the other hand, isothermal amplification guides nucleic acid amplification through the interaction of multiple reaction enzymes, omitting the repeated heating and cooling process of PCR, thereby significantly shortening the nucleic acid detection time.

In recent years, new isothermal amplification detection technology based on microfluidic chips is becoming a research focus for pathogen detection. With the significant advantages of microfluidic chips in fluid drive and automated operation of the detection process, the comprehensive performance of isothermal amplification detection on the microfluidic chip platform can be further improved, and pathogen detection with more powerful functions, higher efficiency, more convenient operation and higher detection sensitivity can be achieved, which has important practical significance for on-site timely detection and real-time epidemic prevention and control of major infectious diseases. In addition, based on the microfluidic platform, high-throughput or multi-target multiple isothermal amplification detection can be achieved, which is conducive to further improving the application of isothermal amplification in on-site timely detection.

technical problem

In order to overcome the defects of the above-mentioned prior art, the technical problem to be solved by the present invention is to provide a constant temperature detection kit and a detection method for detecting pathogens, which has strong specificity, high sensitivity and short detection time. The detection kit and method are for non-diagnostic purposes.

Technical Solutions

In order to solve the above technical problems, the technical solution adopted by the present invention is: a constant temperature detection kit for detecting pathogens, comprising an amplification system, wherein the amplification system comprises a primer set, and the primer set is any one of an African swine fever virus primer set, a porcine circovirus primer set, a pseudorabies virus primer set, a porcine parvovirus primer set or a novel coronavirus primer set;

The African swine fever virus primer set consists of 6 primers shown in SEQ ID NO:1~ SEQ ID NO:6; the porcine circovirus primer set consists of 6 primers shown in SEQ ID NO:7~ SEQ ID NO:12; the pseudorabies virus primer set consists of 6 primers shown in SEQ ID NO:13~ SEQ ID NO:18; the porcine parvovirus primer set consists of 6 primers shown in SEQ ID NO:19~ SEQ ID NO:24; the novel coronavirus primer set consists of 6 primers shown in SEQ ID NO:25~ SEQ ID NO:30 (see Table 1).

Table 1

Another technical solution of the present invention is: a method for detecting pathogens, comprising the following steps:

S1: sampling and preparing sample solution;

S2: inject the sample solution from the injection port of the microfluidic chip, and the sample solution enters the reaction pool with the freeze-dried amplification system, and then seal the pores and injection protrusions of the chip with a sealing film, and insert it into the instrument for reaction;

S3: After the reaction is completed, the instrument automatically interprets the results and displays the test results;

S4: After the test is completed, remove the chip from the instrument and dispose of the chip and reagents as medical waste.

Beneficial Effects

The beneficial effect of the present invention is that: the method for testing viruses of the present invention establishes a rapid detection method based on RT-LAMP amplification technology or LAMP amplification technology, freeze-drying technology and self-absorption discrete microfluidic chip technology, which is to load freeze-dried balls with a virus amplification system in a microfluidic chip reaction pool, the freeze-dried balls contain amplification detection components for the virus, and perform a visual qualitative detection of the pathogen at the endpoint through the H ⁺ ions generated during the amplification process. It is also possible to detect multiple pathogens by replacing different primers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the structure of a microfluidic chip according to a specific embodiment of the present invention;

FIG2 is a schematic structural diagram of a microfluidic chip according to a specific embodiment of the present invention from another angle;

Explanation of reference numerals: 1. chip substrate; 2. first reaction pool; 3. second reaction pool; 4. third reaction pool; 5. fourth reaction pool; 6. air cavity; 7. air hole; 8. injection port; 9. first channel; 10. injection protrusion; 11. second channel.

Embodiments of the present invention

In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in conjunction with the implementation modes and the accompanying drawings.

The key concept of the present invention is that freeze-dried balls with a virus amplification system are loaded into a microfluidic chip reaction pool. The freeze-dried balls contain amplification detection components for the virus, and the H ⁺ ions generated during the amplification process are used to perform a visual qualitative detection of the pathogen at the endpoint.

A constant temperature detection kit for detecting pathogens of the present invention comprises an amplification system, wherein the amplification system comprises a primer set, and the primer set is any one of an African swine fever virus primer set, a porcine circovirus primer set, a pseudorabies virus primer set, a porcine parvovirus primer set or a novel coronavirus primer set;

The African swine fever virus primer set consists of 6 primers shown in SEQ ID NO:1~ SEQ ID NO:6; the porcine circovirus primer set consists of 6 primers shown in SEQ ID NO:7~ SEQ ID NO:12; the pseudorabies virus primer set consists of 6 primers shown in SEQ ID NO:13~ SEQ ID NO:18; the porcine parvovirus primer set consists of 6 primers shown in SEQ ID NO:19~ SEQ ID NO:24; the novel coronavirus primer set consists of 6 primers shown in SEQ ID NO:25~ SEQ ID NO:30.

From the above description, it can be seen that the beneficial effect of the present invention is that the primer sequence designed by the kit of the present invention includes a pair of inner primers (FIP and BIP), a pair of outer primers (F3 and B3) and a pair of loop primers (LF and LB). The six primers target 8 gene sites of the N gene, and the detection of multiple viruses can be achieved by replacing different primers. Among them, the new coronavirus primer set uses the N gene of SARS-COV-2 as a template for LAMP primer design; the African swine fever virus primer set is designed based on the conserved gene B646L encoding the viral protein P72 as a template.

Furthermore, the primer set accounts for 4% to 20% of the total volume of the amplification system.

Furthermore, the primer contents in the primer set are shown in Table 2.

Table 2

It can be seen from the above description that the above ratio is a specific ratio, and changes in the primer content and ratio will affect the specificity of the reaction system and may produce primer dimers.

Furthermore, the amplification system also includes 5x buffer, dye, deoxyribonucleoside triphosphate, Bst DNA polymerase and RNase inhibitor.

From the above description, it can be seen that compared with the common 2x buffer, the solid content of 5x buffer added to the freeze-drying system is higher, which can shorten the freeze-drying time of the subsequent amplification system and improve the stability of the reagent after freeze-drying. The enzyme used in the present invention has stable performance and high titer. After freeze-drying after adding to the reaction system, there is no obvious loss of enzyme activity; the enzyme after freeze-drying is in a dry powder state in the system, and the spatial conformation is stable and not easy to change, so it can also maintain activity at room temperature.

Furthermore, when the primer set is a novel coronavirus primer set, the amplification system also includes reverse transcriptase.

From the above description, it can be seen that when the primer set is a novel coronavirus primer set, the present invention adopts RT-LAMP amplification technology, and when it is other primer sets, LAMP technology is adopted. LAMP (loop-mediated isothermal amplification) amplification technology is a sensitive constant temperature nucleic acid amplification method, which uses Bst DNA polymerase with chain displacement activity and 4-6 primers designed for 6 sites in the region to be amplified for DNA amplification. RT-LAMP is based on LAMP and adds reverse transcriptase, which can reverse transcribe RNA into cDNA and then amplify it directly, thereby expanding the template range from DNA to RNA. LAMP and RT-LAMP technology have the advantages of low equipment requirements, high specificity, and simple operation (providing a constant temperature of 60°C-65°C).

Furthermore, the component contents of the amplification system are shown in Table 3.

table 3

From the above description, we can know that the content of 5x buffer and Bst DNA polymerase will affect the amplification efficiency, and the change in content may cause the system to not amplify; the content of dye will cause the color of the system to not change or change insignificantly before and after the reaction; the content of deoxyribonucleoside triphosphate and primer group will cause the system to not amplify or non-specific amplification, and produce primer dimers and other problems. The content of RNase inhibitor will inhibit the amplification of the system, and the content of RNase inhibitor will cause contamination.

Furthermore, the 5x buffer includes KCl solution, Mg ²⁺ solution Tris-HCl solution and Tween 20.

From the above description, it can be seen that 5x buffer and other components in the amplification system do not contain glycerol and can be directly freeze-dried without adjusting the components.

Furthermore, the component contents of 5x buffer are shown in Table 4.

Table 4

From the above description, it can be seen that excessive content of potassium chloride and magnesium ions leads to reduced amplification efficiency, while insufficient content inhibits amplification, both of which affect the amplification efficiency of the final reaction system; the content of Tris-HCl solution affects the color change of the system.

Furthermore, the pH of the Tris-HCl solution is 9.0.

From the above description, it can be seen that a high pH value will make the color change of the system before and after the reaction less obvious, while a low pH value will cause the system to not change color.

Furthermore, the dye includes 0.1% to 0.5% by mass of Evans blue and 0.1% to 1% by mass of phenol red.

From the above description, it can be seen that when using Evans blue and phenol red composite dye, the color of the system before the reaction of the positive sample is purple-black, and the color of the system after the reaction is yellow-green. The color of the system before and after the reaction of the negative sample is purple-black. After the reaction, the colors of the negative and positive test results are clearly distinguished, which is easier to identify than the common blue-purple color change system or the color change system based on calcein, is less likely to be misjudged, and has a high sensitivity of the test results.

Furthermore, a protective agent is included, which includes 10% to 15% by weight of trehalose, 5% to 10% by weight of BSA (bovine serum albumin), and 10% to 20% by weight of mannitol.

From the above description, it can be seen that adding a certain amount of protective agent to the amplification system for freeze-drying can reduce the loss of enzyme activity and improve detection efficiency.

Furthermore, the amplification system and the protective agent are mixed and then freeze-dried to obtain a freeze-dried reagent.

S1: sampling and preparing sample solution;

S2: injecting the sample solution from the injection port of the microfluidic chip, the sample solution enters the reaction pool with the above-mentioned lyophilized reagent, and then sealing the pores and injection protrusions of the chip with a sealing film, and inserting it into the instrument for reaction;

Furthermore, the amplification system includes primers, buffer, dye, deoxyribonucleoside triphosphates, Bst DNA polymerase and RNase inhibitor.

From the above description, it can be seen that the present invention has established a rapid detection method based on visualized LAMP/RT-LAMP amplification technology, freeze-drying technology and self-priming discrete microfluidic chip technology, which is conducive to realizing "sample in-result out" closed automated detection, significantly improving detection efficiency, eliminating the extraction process required by other detection methods (such as conventional PCR or conventional LAMP), greatly shortening the overall detection time, and obtaining results within 40 minutes; it is also conducive to realizing high-throughput, parallel, and even multi-target multiple detection, significantly improving the overall detection level, and can be conveniently used in public health emergencies, and is more suitable for grassroots units.

After being packaged in the reaction pool of the chip, the freeze-dried balls can be stored at room temperature of 2 to 30°C for 6 months, which effectively avoids the problem of reagent failure caused by improper storage during reagent transportation and reduces the cost of transportation and storage. During visual detection, the H ⁺ ions generated during the LAMP amplification process can be used to perform qualitative endpoint detection of pathogens. When the test is positive, the color of the amplification detection solution will change from purple-black to green; when the test is negative or it is an original system without amplification, the amplification detection solution should be purple-black.

Please refer to Figures 1 and 2 for another technical solution of the present invention: a microfluidic chip includes a chip substrate, on which a first reaction pool, a second reaction pool, a third reaction pool, a fourth reaction pool, an injection mechanism and four cylindrical air cavities are provided; the injection mechanism is respectively connected to the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool; the four air cavities are respectively arranged in one-to-one correspondence with and connected to the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool, an air hole is provided at one end of the air cavity, one side of the air hole passes through the chip substrate, and the diameter of the air hole is smaller than the diameter of the air cavity.

The above-mentioned kit also includes the above-mentioned microfluidic chip.

Furthermore, the chip substrate is made of polydimethylsiloxane.

From the above description, it can be seen that the beneficial effects of the present invention are: the microfluidic chip can be loaded with a variety of biological reagents, including freeze-dried reagents, and the reagents and the chip can be stored at room temperature, effectively avoiding problems such as reagent expiration.

The microfluidic chip no longer needs to connect the chip and pipelines, and the chip substrate contains 4 independent reaction pools, which are connected to the sample inlet and adopt a "no sample outlet design", which effectively avoids the pollution of the environment by the waste liquid after the reaction; the microfluidic chip designed in this scheme is conducive to the realization of "sample in-result out" closed automatic detection, which significantly improves the detection efficiency; on the other hand, it is conducive to the realization of high-throughput, parallel, and even multi-target multiple detection, which significantly improves the overall detection level.

Furthermore, the sample injection mechanism includes a sample injection protrusion and a sample injection port. The sample injection protrusion is arranged on the side of the chip substrate where the pores are not opened. The sample injection protrusion is located directly above the sample injection port. The end of the sample injection protrusion away from the sample injection port is recessed inward to form a cylindrical sample addition cavity. The sample injection port is connected to the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool through the first channel respectively, and the sample injection port connects the sample addition cavity and the first channel.

From the above description, it can be seen that by providing the sample injection protrusion and the sample addition cavity, the sample is injected into the sample addition cavity using a pipette/dropper, which can avoid splashing during injection. By designing the first channel inside the chip substrate, there is no need to connect the chip and the pipeline, which is conducive to realizing the "sample in-result out" closed automatic detection and significantly improving the detection efficiency.

Furthermore, the first reaction tank is circular in shape, the second reaction tank is a regular pentagon in shape, the third reaction tank is a regular octagon in shape, and the fourth reaction tank is a regular hexagon in shape.

From the above description, it can be seen that by setting the four reaction pools into different shapes, if the freeze-dried reagents for internal standards and the freeze-dried reagents for detection are placed in different reaction pools respectively, they can be distinguished according to the shapes of the reaction pools; when the chip is used for multiple target/item detection, the detection targets/detection items can be distinguished by shape.

Furthermore, it also includes a sealing film.

As can be seen from the above description, the sealing film is used to seal the entire microfluidic chip, specifically, to seal the injection protrusions and pores to prevent the sample solution from overflowing.

Furthermore, the four air cavities are connected to the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool respectively through the second channels.

From the above description, it can be seen that by designing a second channel inside the chip substrate, there is no need to connect the chip and the pipeline, which is conducive to realizing "sample in-result out" closed automated detection and significantly improving detection efficiency.

Embodiment 1 of the present invention is:

A virus detection kit of the present invention comprises an amplification system and a protective agent, wherein the amplification system comprises an African swine fever virus primer set, a 5x buffer, a dye, a deoxyribonucleoside triphosphate, a Bst DNA polymerase, and an RNase inhibitor. The 5x buffer is composed of a KCl solution, a Mg2 ⁺ solution, a Tris-HCl solution, and Tween 20. The dye comprises 0.1% by mass of Evans blue and 0.1% by mass of phenol red. The protective agent comprises 10% by mass of trehalose, 5% by mass of BSA (bovine serum albumin), and 10% by mass of mannitol. The amplification system and the protective agent are mixed and freeze-dried to obtain a freeze-dried reagent.

The African swine fever virus primer set consists of 6 primers shown in SEQ ID NO: 1 to SEQ ID NO: 6;

The components of the amplification system are shown in Table 5.

table 5

The actual amount of Tris-HCl solution added in Table 5 is 0.8995 µL, which is approximately 0.9 µL.

The method for detecting common pathogens of pig infection using the above-mentioned constant temperature detection kit comprises the following steps:

S1: sampling and preparing sample solution;

S2: injecting the sample solution from the injection port of the microfluidic chip, the sample solution enters the reaction chamber of the above-mentioned freeze-dried reagent through a thin pipe, and then sealing the chip with a sealing film and inserting it into the instrument for reaction;

Wherein, please refer to FIG. 1 and FIG. 2, the microfluidic chip includes a chip substrate 1 and a sealing film, the sealing film is used to seal the entire microfluidic chip, and the material of the chip substrate 1 is polydimethylsiloxane. The chip substrate 1 is provided with a sample injection mechanism, a first reaction pool 2, a second reaction pool 3, a third reaction pool 4, a fourth reaction pool 5, a first channel 9, a second channel 11 and four cylindrical air cavities 6, one end of the air cavity 6 is provided with an air hole 7, one side of the air hole 7 passes through the chip substrate 1, and the diameter of the air hole 7 is smaller than the diameter of the air cavity 6. The shape of the first reaction pool 2 is circular, the shape of the second reaction pool 3 is a regular pentagon, the shape of the third reaction pool 4 is a regular octagon, and the shape of the fourth reaction pool 5 is a regular hexagon. The geometric center of the first reaction pool 2, the geometric center of the second reaction pool 3, the geometric center of the third reaction pool 4 and the geometric center of the fourth reaction pool 5 are connected to form a square.

The injection mechanism includes an injection protrusion 10 and an injection port 8. The injection protrusion 10 is arranged on the side of the chip substrate 1 where the air hole 7 is not opened. The injection protrusion 10 is located directly above the injection port 8. The end of the injection protrusion away from the injection port is recessed inward to form a cylindrical injection cavity. The injection port 8 is respectively connected to the first reaction pool 2, the second reaction pool 3, the third reaction pool 4 and the fourth reaction pool 5 through the first channel 9. The injection cavity is connected to the first channel through the injection port 8.

The four air cavities 6 are respectively arranged in one-to-one correspondence with the first reaction pool 2 , the second reaction pool 3 , the third reaction pool 4 and the fourth reaction pool 5 and are connected through the second channel 11 .

The parameter settings of the freeze dryer for the freeze-drying amplification system are shown in Table 6:

Table 6

Table 7

Embodiment 2 of the present invention is:

The difference between Example 2 and Example 1 is that: the primer set is a porcine circovirus primer set, and the porcine circovirus primer set consists of 6 primers shown in SEQ ID NO: 7 to SEQ ID NO: 12; the dye includes 0.5% by mass of Evans blue and 1% by mass of phenol red. The protective agent includes 15% by mass of trehalose, 10% by mass of BSA (bovine serum albumin) and 20% by mass of mannitol.

The components of the amplification system are shown in Table 8.

Table 8

The actual amount of Mg ²⁺ solution added in Table 8 is 0.9999 µL, which is approximately 1.0 µL.

Embodiment 3 of the present invention is:

The difference between Example 3 and Example 1 is that: the primer set is a pseudorabies virus primer set, and the pseudorabies virus primer set consists of 6 primers shown in SEQ ID NO: 13 to SEQ ID NO: 18; the dye includes 0.3% by mass of Evans blue and 0.3% by mass of phenol red. The protective agent includes 12% by mass of trehalose, 8% by mass of BSA (bovine serum albumin) and 15% by mass of mannitol.

The components of the amplification system are shown in Table 9.

Table 9

The actual amount of KCl solution added in Table 9 is 2.4993 µL, which is about 2.5 µL.

Embodiment 4 of the present invention is:

The difference between Example 4 and Example 1 is that the primer set is a porcine parvovirus primer set, and the porcine parvovirus primer set consists of 6 primers shown in SEQ ID NO:19 to SEQ ID NO:24.

Embodiment 5 of the present invention is:

The difference between Example 5 and Example 1 is that the primer set is a novel coronavirus primer set, and the novel coronavirus primer set consists of 6 primers shown in SEQ ID NO:25 to SEQ ID NO:30. Each 25 µL amplification system also includes 0.1 µL of reverse transcriptase at a concentration of 200U/µL.

Embodiment 6 of the present invention is:

The difference between Example 6 and Example 5 is that each 25 μL of the amplification system further includes 0.5 μL of reverse transcriptase at a concentration of 200 U/μL.

Embodiment 7 of the present invention is:

The difference between Example 7 and Example 5 is that each 25 μL of the amplification system further includes 1.0 μL of reverse transcriptase at a concentration of 200 U/μL.

In summary, the present invention has established a rapid detection method based on visualized LAMP/RT-LAMP amplification technology, freeze-drying technology and self-priming discrete microfluidic chip technology, which is conducive to realizing "sample in-result out" closed automated detection, significantly improving detection efficiency, eliminating the extraction process required by other detection methods (such as conventional PCR or conventional LAMP), greatly shortening the overall detection time, and obtaining results within 40 minutes; it is also conducive to realizing high-throughput, parallel, and even multi-target multiple detection, significantly improving the overall detection level, and can be conveniently used in public health emergencies, and is more suitable for grassroots units.

During use, it is only necessary to inject the sample solution into the injection protrusion 10, and the sample to be tested flows to the injection port 8 through the injection protrusion 10 on the chip substrate 1. The surface tension of the liquid and the hydrophilic material of the chip produce a siphon effect, so that the sample solution in the injection port 8 enters the reaction pool through the first channel 9. At this time, the gas in the reaction pool passes through the second channel 11 and the air cavity 6 in turn and is discharged from the air hole 7. No other external force is required, and no injection pump is required. After the sample is added, the air hole 7 and the injection protrusion 10 are sealed with a sealing film, and the instrument is inserted for reaction. During the amplification reaction, the detector inputs the received fluorescence signal into the computer, and the supporting software will process the received signal accordingly and draw it into a real-time curve. After the detection is completed, the result is automatically interpreted and displayed.

The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

A constant temperature detection kit for detecting pathogens, characterized in that it comprises an amplification system, wherein the amplification system comprises a primer set, and the primer set is any one of an African swine fever virus primer set, a porcine circovirus primer set, a pseudorabies virus primer set, a porcine parvovirus primer set, or a novel coronavirus primer set;

The African swine fever virus primer set consists of 6 primers shown in SEQ ID NO:1~ SEQ ID NO:6; the porcine circovirus primer set consists of 6 primers shown in SEQ ID NO:7~ SEQ ID NO:12; the pseudorabies virus primer set consists of 6 primers shown in SEQ ID NO:13~ SEQ ID NO:18; the porcine parvovirus primer set consists of 6 primers shown in SEQ ID NO:19~ SEQ ID NO:24; the novel coronavirus primer set consists of 6 primers shown in SEQ ID NO:25~ SEQ ID NO:30.
The isothermal detection kit for detecting pathogens according to claim 1 is characterized in that the amplification system also includes 5x buffer, dye, deoxyribonucleoside triphosphate, Bst DNA polymerase and RNase inhibitor.
The constant temperature detection kit for detecting pathogens according to claim 1, characterized in that when the primer set is a novel coronavirus primer set, the amplification system also includes a reverse transcriptase.
The constant temperature detection kit for detecting pathogens according to claim 2, characterized in that the 5x buffer comprises KCl solution, Mg2 + solution Tris-HCl solution and Tween 20.
The constant temperature detection kit for detecting pathogens according to claim 4, characterized in that the pH of the Tris-HCl solution is 9.0.
The constant temperature detection kit for detecting pathogens according to claim 2, characterized in that the dye comprises 0.1% to 0.5% by mass of Evans blue and 0.1% to 1% by mass of phenol red.
The constant temperature detection kit for detecting pathogens according to claim 1 is characterized in that it also includes a protective agent, wherein the protective agent includes 10% to 15% by mass of trehalose, 5% to 10% by mass of BSA, and 10% to 20% by mass of mannitol.
The constant temperature detection kit for detecting pathogens according to claim 7 is characterized in that the amplification system and the protective agent are mixed and then freeze-dried to obtain a freeze-dried reagent.
A method for detecting a pathogen, characterized in that it comprises the following steps:

S1: sampling and preparing sample solution;

S2: injecting the sample solution from the injection port of the microfluidic chip, the sample solution enters the reaction pool containing the lyophilized reagent according to claim 8, and then sealing the pores and injection protrusions of the chip with a sealing film, and inserting the chip into the instrument for reaction;

S3: After the reaction is completed, the instrument automatically interprets the results and displays the test results;

S4: After the test is completed, remove the chip from the instrument and dispose of the chip and reagents as medical waste.
The detection method for detecting pathogens according to claim 9 is characterized in that the microfluidic chip includes a chip substrate, and the chip substrate is provided with a first reaction pool, a second reaction pool, a third reaction pool, a fourth reaction pool, a sample injection mechanism and four cylindrical air cavities; the sample injection mechanism is respectively connected to the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool; the four air cavities are respectively arranged in a one-to-one correspondence with the first reaction pool, the second reaction pool, the third reaction pool and the fourth reaction pool and are connected, one end of the air cavity is provided with an air hole, one side of the air hole passes through the chip substrate, and the diameter of the air hole is smaller than the diameter of the air cavity.