CN111690510B

CN111690510B - Pathogenic bacteria detection reactor and preparation and application methods thereof

Info

Publication number: CN111690510B
Application number: CN202010528203.3A
Authority: CN
Inventors: 周麟; 葛玉卿; 毛红菊; 赵建龙; 杨宏波; 张欣
Original assignee: Shanghai Prospective Innovation Research Institute Co ltd; Shanghai Institute of Microsystem and Information Technology of CAS
Current assignee: Shanghai Prospective Innovation Research Institute Co ltd; Shanghai Institute of Microsystem and Information Technology of CAS
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2023-07-21
Anticipated expiration: 2040-06-11
Also published as: CN111690510A

Abstract

The invention discloses a pathogenic bacteria detection reactor and a preparation and application method thereof, relating to the technical field of pathogenic bacteria detection, and comprising the following steps: two layers of films are sealed to form a cracking zone, a mixing zone and a reaction zone which are vertically arranged; one side of the cracking zone is separated from the mixing zone by a packaging isolation zone, and the other side of the cracking zone is provided with a sample inlet; the reaction buffer zone is respectively communicated with the mixing zone and the reaction zone; the reaction zone comprises at least one first reaction zone for embedding an internal reference target and at least one second reaction zone for embedding a test target; the first reaction and the second reaction junction each have a reaction member comprising at least one reaction cell; the problems of complex structure, complex operation and higher cost of a pathogen detection microfluidic chip in the prior art are solved.

Description

Pathogenic bacteria detection reactor and preparation and application methods thereof

Technical Field

The invention relates to the technical field of pathogenic bacteria detection, in particular to a pathogenic bacteria detection reactor and a preparation and detection method thereof.

Background

According to the world health organization report, the cases of diarrhea are up to 1.5 hundred million worldwide, wherein 70% of cases are related to food pollution caused by various pathogenic microorganisms, and according to the latest data, microorganisms are the main pathogens of food-borne diseases and account for approximately half of the total number, so that the rapid detection of pathogenic bacteria has been directly related to national security, social and economic development and social folks.

The method based on nucleic acid amplification is an effective and accurate pathogen identification method, but the method requires tedious operations (including cracking, nucleic acid extraction, amplification and even sequencing), and the micro-fluidic chip technology is a technology of integrating basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes onto one micron-scale chip and automatically completing the whole analysis process, and integrating the steps into the same micro-fluidic chip is an urgent requirement for the protection of infectious pathogenic bacteria, sanitary epidemic prevention inspection and on-site rapid detection of food safety at present.

However, in order to meet the demands of integration, tightness and biocompatibility of the steps, most of the conventional pathogen detection microfluidic chips are complex in structure, and are especially not suitable for detection application of pathogen extraction-free purification reagent systems, and are complex in operation and high in cost.

Disclosure of Invention

The invention aims to provide a pathogenic bacteria detection reactor and a preparation and detection method thereof, which are used for solving the problems of complex structure of a pathogenic bacteria detection microfluidic chip, and particularly inapplicability to detection application of a pathogenic bacteria extraction-free purification reagent system, thus having complicated operation and higher cost.

To achieve the above object, the present invention provides a pathogen detection reactor comprising: two layers of films are sealed to form a cracking zone, a mixing zone and a reaction zone which are vertically arranged and communicated in sequence;

one side of the cracking zone is separated from the mixing zone by a packaging isolation zone, and the other side of the cracking zone is provided with a sample inlet;

the reaction zone comprises at least one first reaction zone for embedding an internal reference target and at least one second reaction zone for embedding a test target;

the first reaction and the second reaction each have a reaction piece comprising at least one reaction cell.

Further, at least one reaction buffer area is arranged between the reaction area and the mixing area, and control valves are arranged on pipelines communicated with the reaction area and the mixing area in the reaction buffer area.

Further, the first reaction zone and the second reaction zone are respectively communicated with the mixing zone through independent reaction buffer zones.

Further, the packaging isolation belt is a hot-pressing narrow stripe and is used for forming at least one mixing channel after being extruded from the cracking zone or the mixing zone.

Further, the film is made of a transparent flexible polymer material, and the polymer material is one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer material.

Further, two second reaction areas for embedding the test targets are arranged, and the first reaction areas and the second reaction areas are arranged in parallel.

Further, the reaction zone also has a plurality of redundant liquid zones; each redundant liquid zone is connected with the first reaction zone and the second reaction zone respectively;

one end of the redundant liquid area is communicated with one side of the reaction cavity, which is close to the reaction buffer area.

Further, the reaction member includes a structural plate having at least one through hole;

the sealing film layer is arranged on one side of the structural plate, and the film layer with the small holes is arranged on the other side of the structural plate;

the through holes and the film layers on the two sides form a reaction tank.

Furthermore, the reaction tank is pre-embedded with a primer for PCR reaction, DNTP and enzyme required by the reaction.

In order to achieve the above object, the present invention further provides a method for preparing a pathogen detection reactor, for preparing any one of the pathogen detection reactors, comprising: and (3) aligning two layers of films to ensure that the cracking zone, the mixing zone and the reaction zone are communicated and then sealed.

Further, the sealing mode adopts one or more modes of laser welding, hot press sealing, high-strength chemical glue bonding or ultrasonic welding.

In order to achieve the above object, the present invention also provides a method for applying the reactor for detecting pathogenic bacteria, wherein the method for applying any one of the above reactors for detecting pathogenic bacteria comprises the following steps:

step one: the method comprises the steps of (1) enabling a test sample to enter a cracking zone of a pre-stored cell lysate through a sample inlet, and heating the cracking zone to crack the test sample;

step two: extruding and breaking through the packaging isolation belt, so that the cracked test sample enters the mixing area and controls the test sample to flow back and forth in the mixing area and the cracking area, and the test sample releases nucleic acid molecules to obtain the test sample with the nucleic acid molecules;

step three: dispersing the test sample with the nucleic acid molecules into a first reaction zone and a second reaction zone, so that the test sample reacts in a reaction tank;

step four: detecting whether each reaction cell has a specific target.

Further, in the third step, the liquid overflowing the reaction tank is controlled to enter the redundant liquid area before the test sample reacts in the reaction tank.

Furthermore, the test sample in the third step is subjected to isothermal amplification or thermocycling amplification reaction with the primer of the PCR reaction pre-buried in the reaction tank, DNTP and enzyme required by the reaction under the preset temperature environment.

In the fourth step, each reaction cell is excited by a fluorescent light source, the fluorescent intensity in each reaction cell is detected, and whether a specific target exists is judged based on the fluorescent intensity in each reaction cell.

According to the pathogen detection reactor and the preparation and detection methods thereof, provided by the invention, the cracking zone, the mixing zone and the reaction zone are arranged up and down, the test sample enters the cracking zone to be completely cracked and then directly enters the mixing zone to be fully mixed and cracked, and then the test sample is subjected to amplification reaction in the reaction zone, so that the problems that a pathogen detection microfluidic chip in the prior art is complex in structure, complex in operation and high in cost, and is not suitable for detection application of a pathogen extraction-free purification reagent system are solved.

Drawings

FIG. 1 is a schematic view of a pathogenic bacteria detecting reactor according to a first embodiment of the present invention;

FIG. 2 is a schematic view showing the structure of a sealing portion of a pathogenic bacteria detecting reactor according to a first embodiment of the present invention;

FIG. 3 is a schematic structural view of a reaction plate in a first embodiment of a detecting method of a pathogenic bacteria detecting reactor according to the present invention;

FIG. 4 is a flow chart of a third embodiment of a method of detecting a pathogenic bacteria detection reactor according to the present invention;

fig. 5 is a schematic structural diagram of a pneumatic control piston according to an example of a third embodiment of a detecting method of a pathogenic bacteria detecting reactor of the present invention.

Reference numerals:

1. a cleavage zone; 12. a closing cap; 2. a mixing zone; 3. a reaction zone; 31. a reaction buffer zone; 32. a reaction zone; 33. a redundant liquid zone; 322. a reaction tank; 4. packaging the isolation belt; 5. a control valve; 61. a thin film layer with small holes; 62. a structural panel; 63. a closed film layer; a-sealing part.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, a "first" element may be termed a "second" element without departing from the teachings of the present embodiment.

It should be noted that the words "front", "back", "left", "right", "upper" and "lower" used in the following description refer to directions in the drawings, and the words "bottom" and "top", "inner" and "outer" refer to directions toward or away from, respectively, the geometric center of a particular component.

It should also be noted that the pathogen detection reactor described in the following embodiments is mainly used for detecting pathogen hands-free purification reagent systems.

Embodiment one:

the present application provides a pathogen detection reactor, referring to fig. 1 and 2, comprising: two layers of films are sealed to form a cracking area which is vertically arranged in sequence and communicated and is used for preassembling a cracking solution, a mixing area for obtaining nucleic acid molecules and a reaction area for carrying out nucleic acid amplification and detection; the cracking zone, the mixing zone and the reaction are all arranged in sequence from top to bottom.

In the above embodiment, the reactor is integrally configured as a reaction chamber structure that is distributed vertically, a region for sample splitting is provided above the reactor, a region for sample mixing is provided in the middle of the reactor, a region for sample reaction is provided below the reactor, and such a vertical structural design enables the internal fluid flow to be controlled simultaneously by using external control force and gravity, so that the flow process is more efficient and rapid, the liquid loss in each process is reduced, and the operation can be performed by means of a pneumatic control piston, so that the sample retention time in each region is controlled.

One side of the cracking zone is separated from the mixing zone by a packaging isolation belt, and the other side of the cracking zone is provided with a sample inlet with a sealing cap; in a preferred embodiment, the packaging isolation belt is a hot-pressing narrow strip, which is used for forming at least one mixing channel after being extruded from the cracking zone or the mixing zone, the bent hot-pressing narrow strip can be used as an isolation belt for pre-storing reagents in the reactor or an isolation belt for two different reaction parts, when the reaction is needed, the isolation belt can be extruded to form a plurality of mixing channels by virtue of a pneumatic control piston through extrusion, two physically isolated areas are communicated, and the structure can be used as an isolation switch for a reagent pre-storing area, so that the manufacturing and the operation are simple and convenient, and the space efficiency of pre-embedding the reagents in the reactor can be effectively improved.

The reaction area comprises a first reaction area for embedding the memory targets and at least one second reaction area for embedding the test targets, and the embedded multiple test targets can be embedded into the same test target or different test targets. The first reaction zone and the second reaction zone are both provided with reaction pieces comprising at least one reaction tank, in the specific implementation mode, one first reaction zone and two second reaction zones are arranged, two second reaction zones for pre-burying test targets are arranged, the first reaction zone and each second reaction zone are arranged in parallel, three reaction buffer zones are arranged to serve as independent reaction zones for different detection targets, and in the use process, pneumatic control pistons are matched, so that the reaction processes of the targets are mutually independent, mutual pollution and interference between the different targets are avoided, and in the specific implementation mode, the first reaction zone and the two second reaction zones are arranged in parallel, and the chip space utilization rate is effectively improved.

In the above embodiment, as shown in fig. 3, the reaction member includes a structural plate with at least one through hole; the through holes and the film layers on two sides form a reaction tank; in this embodiment, three reaction tanks are disposed on each structural plate, and the reaction tanks are pre-embedded with a primer for PCR reaction, DNTP and an enzyme required for reaction, for assisting in performing an amplification reaction on a nucleic acid molecule extracted from a test sample after a liquid enters the reaction tanks, where the above arrangement manner can provide three reaction chambers of the same size, and ensure that the same target detection reaction can be repeated 3 times at the same time, and provide a powerful guarantee as to the accuracy of the detection result, so as to reduce the situation that the detection result is inaccurate due to contingency.

In a preferred embodiment, a reaction buffer area is arranged between the reaction area and the mixing area, the purpose of the reaction buffer area is mainly to control the speed of liquid entering the first reaction area or the second reaction area from the mixing area, so that the influence on the reaction process caused by a large amount of liquid directly rushing into the first reaction area or the second reaction area under the assistance of gravity due to vertical arrangement is reduced, even the situation of damaging a film is caused, the control valves are arranged on pipelines of the first reaction buffer area, the reaction area and the mixing area, and the control of the liquid flow direction is realized by the aid of the control valves and the separation part auxiliary pneumatic control piston in the reaction process; the first reaction zone and the second reaction zone are respectively communicated with the mixing zone through independent reaction buffer zones.

In a preferred embodiment, the reaction zone also has a plurality of redundant liquid zones; each redundant liquid zone is connected with the first reaction zone and the second reaction zone respectively; in this embodiment, one end of the redundant liquid area is communicated with one side of the reaction cavity, which is close to the reaction buffer area, and one end of the redundant liquid area is communicated with one side of the first reaction area or the second reaction area, which is close to the reaction buffer area, and the other end of the redundant liquid area is bent and extended to the outer side of the bottom of the first reaction area or the second reaction area, each redundant liquid area is in a reverse L-shaped arrangement, and the redundant liquid area is used for enabling redundant liquid after entering the reaction cavity to enter the redundant liquid area, so that the influence of the redundant liquid on the amplification reaction process is reduced.

Through the arrangement mode, each first reaction zone or each second reaction zone and each corresponding reaction buffer and redundant liquid zone form an independent reaction space between the three zones, and each independent reaction space is used for embedding different targets (internal reference targets or test targets), so that independent testing of each target is further facilitated, mutual interference in the testing process is reduced, and accuracy of a testing result is improved.

In a preferred embodiment, the membrane is a transparent flexible polymeric material, the polymeric material being one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer, the flexible material being primarily adapted to facilitate control of the flow of liquid during use by application of pressure to the lysing, mixing or reaction zone, and illustratively pneumatic pressure, to allow the flow of sample and reagent mixtures in the various channels, the transparent material being adapted to facilitate clear observation of the flow of liquid by an operator for better control of the reaction process. The film reactor prepared by the material has high temperature control efficiency, and the temperature difference between the reagent in the chip and the instrument temperature control module is reduced due to the temperature circulation of the nucleic acid amplification chip, so that the temperature can be well controlled in the cracking and amplification process; meanwhile, the biological compatibility is good, the biological reagent used in the nucleic acid amplification reaction process generally comprises components such as a primer, enzyme and the like, and the chip materials are required to be compatible with the components so as to ensure the amplification efficiency; in addition, the sealing performance is better, and compared with the common plate type in the prior art, the plate type heat-resistant plate has certain flexibility, and the damage caused by external action in the reaction process is reduced.

The utility model provides a pathogenic bacteria detection reactor, set up the schizolysis district through upper and lower formula, the mode of mixed district and reaction zone, test sample gets into direct entering mixed district after the schizolysis is accomplished to the schizolysis district and makes test sample intensive mixing schizolysis, then make test sample enter into first reaction zone or second reaction zone through reaction buffer area and carry out the amplification reaction, can improve the space utilization of chip and be favorable to controlling test sample's reaction process again, pathogen detection microfluidic chip structure is complicated among the prior art has been solved, be particularly useful for the pathogen and exempt from to draw the detection application of purification reagent system, therefore complex operation and the higher problem of cost, through the reactor of above-mentioned simple design, realize pathogenic bacteria detection through the detection target of pre-buried in reaction zone, and convenient operation, follow-up preparation technology is also simpler.

Embodiment two:

the application provides a preparation method of a pathogenic bacteria detection reactor, which is used for preparing any pathogenic bacteria detection reactor in the first embodiment, and comprises the following steps: the splitting area, the mixing area and the reaction area are communicated and then sealed by adopting two layers of films for alignment (the sealing part can be referred to as fig. 2).

In the above embodiment, each area is reserved by the film and is directly sealed after alignment, and the film can be directly integrally pressed, so that compared with the common injection molding structure in the prior art, the film has simpler structure and processing process, and the processing cost can be greatly reduced.

In a preferred embodiment, the sealing mode adopts one or more modes of laser welding, hot press sealing, high-strength chemical glue bonding or ultrasonic welding, and can be processed by one or more modes according to actual production conditions.

Embodiment III:

the application provides a detection method using a pathogenic bacteria detection reactor, referring to fig. 4, an application embodiment of the pathogenic bacteria detection reactor can be matched with a control device for use, and includes the following steps:

in the above embodiment, by way of example, the test sample may be introduced into the lysis zone by means of a matched injection device, and the temperature control is more convenient when the lysis zone is heated due to the lower film thickness.

Step two: the extrusion breaks through the packaging isolation belt, so that the cracked test sample enters the mixing area and controls the test sample to flow back and forth in the mixing area and the cracking area, and the test sample releases nucleic acid molecules to obtain the test sample with the nucleic acid molecules.

Specifically, extrusion breaks through the packaging isolation belt, and when the cracked test sample enters the mixing area and is extruded, the test sample flows back and forth in the mixing area and the cracking area, can be realized by adopting a pneumatic piston, and the flow direction of the liquid is controlled by giving a certain pressure to the cracking area and the mixing area.

Step three: and opening the control valve, dispersing the test sample with the nucleic acid molecules into the first reaction zone or the second reaction zone, closing the control valve when the test sample in the first reaction zone and the second reaction zone is full, and enabling the test sample to react in the reaction tank.

Specifically, a primer for PCR reaction, DNTP (deoxyribonucleoside triphosphate) and an enzyme required for the reaction are pre-buried in the reaction tank so as to satisfy the reaction conditions for completing the nucleic acid amplification in the reaction tank. And in the third step, after the control valve is closed, the first reaction area or the second reaction area is extruded, so that the sample overflowed from each reaction tank enters a redundant liquid area, and the influence of redundant liquid deposition in the reaction area on the nucleic acid amplification process and the accuracy of the subsequent detection result are reduced.

Step four: and waiting for the reaction to finish, and detecting whether a specific target exists in each reaction tank.

In the above embodiment, the fluorescence intensity in each reaction cell is excited and detected by a fluorescent light source, and whether or not a specific target is present is determined based on the fluorescence intensity. By way of example, but not limitation, the fluorescence light source excitation can be implemented by using a matched detection instrument, or by using other excitation modes in the prior art, and by setting a fluorescence intensity threshold value and detecting the fluorescence intensity value in each reaction cell to determine whether a specific target exists, the accuracy of the detection result can be improved, and other detection modes in the prior art are applicable to the detection result besides fluorescence excitation.

Taking specific example of detecting a pathogen in use, referring to fig. 1 and 5, the method specifically includes the following steps:

embedding a lysate in a lysis cavity, and embedding a primer for PCR reaction, DNTP (deoxynucleoside triphosphate) and enzyme required by the reaction in a reaction tank; the detected internal reference can be pre-buried yeast or phage, and the pre-buried detection target can be one or more of staphylococcus aureus, escherichia coli o157, listeria, campylobacter and salmonella, and each reaction cavity is supposed to be extruded or extruded under the control of a pneumatic piston.

In the following specific steps, the cracking zone 1 is correspondingly provided with a pneumatic control piston 11, and the mixing zone 2 is correspondingly provided with a pneumatic control piston 21; the reaction buffer zone 31 is correspondingly provided with a pneumatic piston 311; the reaction zone 32 is correspondingly provided with a pneumatic piston 321; the pneumatic pistons which are correspondingly arranged on the reaction parts can give the pressure to the reaction cavities, and the pneumatic pistons are closed to remove the applied pressure.

The sample is injected into the cracking zone of the film reactor through a sample inlet, the sample enters the sample cracking zone 1, pathogenic bacteria or cell cracking liquid is pre-stored in the sample cracking zone 1, the sample is fully cracked through heating and mixing, the sealed isolation of hot-pressing narrow strips is broken through a pneumatic piston 11, the cracked sample is extruded into a sample mixing zone 2 from the sample cracking zone 1, the cracked sample is extruded and mixed back and forth in the sample cracking zone 1 and the sample mixing zone 2 through a pneumatic control piston 11 and a pneumatic control piston 21 to fully crack the sample and release nucleic acid molecules, after the sample is fully mixed, the cracked sample is left in the mixing zone 2 through the pneumatic control piston 11, a control valve between the mixing zone 2 and the reaction buffer zone 31 is opened, the sample with the nucleic acid molecules is dispersedly extruded into the reaction buffer zone 31, when the sample liquid in each reaction buffer zone 31 is full, closing the control valve between the mixing zone 2 and the reaction buffer zone 31 and opening the control valve between the buffer zone 31 and the reaction zone 32 (comprising a first reaction zone and two second reaction zones, not specifically shown in the figure), opening the pneumatic control piston 21, respectively extruding the sample in the reaction buffer zone 31 into the reaction cell 322 in combination with the pneumatic control piston 311, closing the control valve between the buffer zone 31 and the reaction chamber 32 after the sample enters the reaction cell 322, opening the pneumatic control piston 321, respectively extruding redundant liquid into the redundant liquid zone 33 corresponding to each of the first reaction zone or the second reaction zone, performing isothermal amplification or thermal circulation in the reaction cell 322, waiting for the end of the reaction, exciting and detecting the fluorescence intensity in the reaction cell in each reaction cell 322 by a fluorescence light source, wherein the reaction cell 322 in each reaction plate has the same target, after excitation by the fluorescent light source in the reaction cell 322, the fluorescence intensity value in each well is detected to determine whether the corresponding target exists.

The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments.

The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but rather is intended to cover any equivalents of the structures or equivalent processes disclosed herein or in the alternative, which may be employed directly or indirectly in other related arts.

Claims

1. A pathogen detection reactor, comprising:

two layers of films are sealed to form a cracking zone, a mixing zone and a reaction zone which are vertically arranged and communicated in sequence;

the first reaction zone and the second reaction zone each have a reaction member comprising at least one reaction cell;

at least one reaction buffer area is arranged between the reaction area and the mixing area, and control valves are arranged on pipelines communicated with the reaction area and the mixing area;

the first reaction zone and the second reaction zone are respectively communicated with the mixing zone through independent reaction buffer zones;

the packaging isolation belt is a hot-pressing narrow stripe and is used for forming at least one mixing channel after being extruded from the cracking zone or the mixing zone;

the reaction member includes a structural plate with at least one through hole;

the through holes and the film layers on the two sides form a reaction tank.

2. A pathogen detection reactor according to claim 1, wherein:

the film is made of transparent flexible polymer material, and the polymer material is one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer material.

3. A pathogen detection reactor according to claim 1, wherein:

the two second reaction areas for embedding the test targets are arranged, and the first reaction areas and the second reaction areas are arranged in parallel.

4. A pathogen detection reactor according to claim 1, wherein:

the reaction zone also has a plurality of redundant liquid zones; each redundant liquid zone is connected with the first reaction zone and the second reaction zone respectively;

5. A pathogen detection reactor according to claim 1, wherein:

the reaction tank is pre-embedded with a primer for PCR reaction, DNTP and enzyme required by the reaction.

6. A method of preparing a pathogen detection reactor for use in preparing a pathogen detection reactor according to any one of claims 1 to 5, comprising the steps of:

and (3) aligning two layers of films to ensure that the cracking zone, the mixing zone and the reaction zone are communicated and then sealed.

7. The method of claim 6, wherein the step of preparing the reactor comprises:

the sealing mode adopts one or more modes of laser welding, hot-press sealing, high-strength chemical glue bonding or ultrasonic welding.

8. A method for detecting a pathogenic bacteria detection reactor using any one of the above claims 1 to 5, comprising the steps of:

step four: detecting whether each reaction cell has a specific target.

9. The method of claim 8, wherein the step of using the pathogen detection reactor comprises:

in the third step, before the test sample reacts in the reaction tank, controlling the liquid overflowing the reaction tank to enter a redundant liquid area;

the reaction zone also has a plurality of the redundant liquid zones; each redundant liquid zone is connected to a first reaction zone and a second reaction zone, respectively.

10. The method of claim 8, wherein the step of using the pathogen detection reactor comprises:

and step three, carrying out isothermal amplification or thermocycling amplification reaction on the test sample, the primer of the PCR reaction pre-buried in the reaction tank, DNTP and enzyme required by the reaction under the preset temperature environment.

11. The method of claim 8, wherein the step of using the pathogen detection reactor comprises:

and step four, exciting each reaction tank by adopting a fluorescent light source, detecting the fluorescent intensity in each reaction tank, and judging whether a specific target exists or not based on the fluorescent intensity in each reaction tank.