CN111662816A - Micro-fluidic chip-based environmental pathogen rapid detection and early warning system and method - Google Patents

Abstract

The invention discloses a microfluidic chip-based rapid detection and early warning system and method for environmental pathogens. The system includes an enrichment component: used to trap pathogens in the air on a filter membrane through a structural component, and then pass through a nucleic acid-free buffer solution. Dissolve to form enrichment solution; micro-reaction component: used to pump the enrichment solution to the micro-reaction chamber for LAMP amplification, so that the enrichment solution reacts with the reaction system with dye added and the reaction system without dye added to form a qualitative Result; communication component: used to receive quantitative detection results and early warning instructions; remote server calculates precipitation turbidity value and virus concentration according to electrical signals to output quantitative detection results and early warning instructions; early warning component: used to issue sound and/or light alarms Signal. The invention utilizes high-efficiency amplification technology to rapidly detect pathogenic viruses in the air, performs qualitative and quantitative detection, outputs detection results and alarm signals, and realizes early warning of viruses in indoor air.

Description

Rapid detection and early warning system and method for environmental pathogens based on microfluidic chip

technical field

The invention relates to the fields of medicine and biology, in particular to isothermal nucleic acid amplification technology, in particular to a rapid detection and early warning system and method for viral pathogens in indoor environments based on microfluidic chip detection.

Background technique

Pathogenic viruses in the air are an important way of disease transmission, such as hand, foot and mouth, influenza virus, SARS virus, etc. Usually after enrichment by a certain method, the samples are cultured in a special incubator. After the virus has been cultured to a certain stage, it is detected and observed by electron microscopy, or the detection of pathogenic antibodies and antigens by immune technology. These require different professional The scientific research personnel cooperate with different laboratories. The general process is complex and time-consuming. The equipment and devices are numerous and huge, and the initial content of the detection is required to be high. Due to the long process time, many instruments and personnel are involved, and there is also pollution. risks of.

Especially due to the low copy number of viruses in the air, many traditional methods need to enrich the pathogens in the air for a long time. For example, the method of collecting pathogens by trapping the air with membranes often takes more than 24 hours to collect the initial limit of detection. Then use a specific host to culture the virus, usually for 48-72 hours, observe by electron microscope, or judge by combining antigen and antibody detection. During this period, multiple sets of devices are involved: enrichment device, culture device, electron microscope, antibody antigen reagent, etc. The procedures are complicated In addition, it is time-consuming and labor-intensive. Among them, the enrichment device and electron microscope are huge; the observation of pathogens and electron microscopes requires high professionalism of operators, and is not suitable for daily households and ordinary living places.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the shortcomings of the above-mentioned background technology, and propose a microfluidic chip-based rapid detection and early warning system and method for environmental pathogens, which utilizes high-efficiency amplification technology to rapidly detect pathogenic viruses in the air, and Carry out qualitative and quantitative detection, output detection results and alarm signals, and realize early warning of viruses in indoor air.

In order to achieve the above purpose, the microfluidic chip-based rapid detection and early warning system for environmental pathogens designed by the present invention is special in that the system includes an enrichment component, a microreaction component, a communication component and an early warning component;

The enrichment component: used to suck the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured to the nucleic acid-free. The enrichment solution is formed in the buffer;

The micro-reaction component is used to pump the enrichment solution to the microfluidic chip plate for LAMP amplification, that is, to perform amplification of specific pathogen nucleic acid fragments, so that the enrichment solution is respectively mixed with the reaction system with dye added and without dye added. The reaction system reacts, and the qualitative results of the reaction between the enrichment solution and the reaction system with dye added are displayed through a transparent window, and the quantitative results of the precipitation turbidity reaction generated by the reaction between the enrichment solution and the reaction system without dye addition are collected by photoelectric equipment;

The communication component: used to receive the electrical signal collected by the optoelectronic equipment, wirelessly transmit the electrical signal to the remote server, receive the quantitative detection result of the reaction and the early warning instruction sent by the remote server, and send it to the display device and the early warning component; the remote server Calculate the precipitation turbidity value and virus concentration according to the electrical signal, and output the quantitative detection results and early warning instructions according to the virus concentration and pathogen target curve;

The pre-warning component: used to issue a sound and/or light alarm signal according to the pre-warning instruction sent by the communication component.

Further, the enrichment assembly includes an air pump, and the air pump is connected to the air outlet at the top of the enrichment bin through a solenoid valve and a pipeline, and an ultrasonic vibration disc is arranged at the bottom of the enrichment bin. The side of the silo is provided with an air inlet, a liquid inlet and a liquid outlet, and a microporous filter membrane is arranged in the inner cavity of the enrichment silo. The front collecting net tank is covered and fixed, the air inlet is connected with the air inlet device through a solenoid valve, the liquid inlet is connected with the first quantitative liquid injection device and the second quantitative liquid injection device through the electromagnetic valve, and the outlet The liquid port is connected with the micro-reaction component.

Further, the micro-reaction assembly includes an observation window, a microfluidic chip plate, a temperature measurement plate and a temperature control plate arranged in sequence from top to bottom, and the microfluidic chip plate includes a sample injection hole arranged in the center, The bottom of the injection hole is communicated with the output end of the enrichment component, a number of sample flow channels are arranged along the periphery of the injection hole, and a micro-reaction chamber is arranged at the end of each sample flow channel. An observation port is provided at a position corresponding to the observation window and the micro-reaction chamber, and a color comparison card corresponding to the pathogen is provided below the observation port.

Further, the first quantitative injection device and the second quantitative injection device have the same structure, and are provided with a base, a push rod and a cavity, the push rod and the cavity are arranged on the base, and one end of the cavity is connected to the base. The push rod is connected, and the other end is connected with the liquid inlet through a solenoid valve. The cavity of the first quantitative liquid injection device is provided with a nucleic acid-free buffer solution, and the cavity of the second quantitative liquid injection device is free of liquid and stored without nucleic acid. Bacteria and virus-free treated air.

Further, the micro-reaction chambers on the microfluidic chip board are respectively arranged in two areas of qualitative detection and quantitative detection, the micro-reaction chamber located in the qualitative detection area is opposite to the observation port, and the inner cavity of the micro-reaction chamber A dry powdery reagent added with dye is provided, and both ends of the micro-reaction chamber located in the quantitative detection area are respectively provided with an LED emitting end and an LED light receiving end, and the LED emitting end and the LED light receiving end are electrically connected with the communication component. , the inner cavity of the micro-reaction chamber is provided with a dry powder reagent without dye.

Further, the communication component includes an A/D converter, an MCU and a wireless communication module, the A/D converter is used for receiving electrical signals from the LED transmitting end and the LED light receiving end, and the MCU is used for passing the wireless communication module. The electrical signal is wirelessly transmitted to the remote server, and the response quantitative detection result and the early warning instruction sent by the remote server are received through the wireless communication module, and the reaction quantitative detection result and the early warning instruction are sent to the display device and the early warning component.

Further, the microporous filter membrane is a 0.01um polytetrafluoroethylene membrane or a polyvinylidene fluoride membrane with a molecular weight cut-off of 100,000.

Further, the diameter of the upper film rear mesh groove liner and the lower film front collecting mesh groove is a cm, a≥1, with the center as the dot, the radius is 0.1a, 0.2a, 0.3a, 0.4a cm Make circles with a radius of 0.02 cm at the center and on the boundary of each circle.

Further, the microfluidic chip board is made of polydimethylsiloxane polymer.

Further, the enrichment bin, the first quantitative liquid injection device, the microfluidic chip board and the corresponding pipeline are replacement devices after use.

Further, the system further includes a remote mobile terminal, and the remote mobile terminal receives the quantitative detection result of the reaction by connecting to the remote server.

The present invention also proposes a microfluidic chip-based rapid detection and early warning method for environmental pathogens, which is special in that the method includes the following steps:

1) Suction the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured in the nucleic acid-free buffer to form enrichment. liquid;

2) The enrichment solution is injected into the microfluidic chip plate for LAMP amplification, that is, the amplification of specific pathogen nucleic acid fragments;

3) The amplified enrichment solution is introduced into the microfluidic chip plate to react with the pre-installed dry powder reaction system, and the temperature and reaction time of the reaction chamber are controlled. The microreaction chamber is divided into qualitative detection and quantitative detection. In each area, dye is added to the dry powder reaction system in the qualitative detection area, and no dye is added to the dry powder reaction system in the quantitative detection area;

4) Determine the pathogen concentration range by comparing the color of the color card and the reaction solution in the micro-reaction chamber in the qualitative detection area, and calculate the pathogen concentration by collecting the turbidity of the precipitate generated by the reaction between the enrichment solution and the reaction system without dye added by photoelectric equipment. ;

5) Calculate the precipitation turbidity value and the virus concentration according to the precipitation turbidity generated by the reaction between the enrichment solution and the reaction system without adding the dye, and output the quantitative detection result of the reaction according to the standard curve relationship between the virus concentration and the pathogen concentration. When the detection result exceeds the preset value A warning command is issued when the value is set.

Preferably, the components of the nucleic acid-free buffer in the step 1) are 200 mM Tris-HCL (pH 8.8 at 25 degrees Celsius), 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCL, 20 mM MgSO ₄ , 1% (v/ v) Tritonx-100.

Preferably, the method for dissolving pathogens in the nucleic acid-free buffer in the step 1) is to submerge the nucleic acid-free buffer to the filter membrane that retains the pathogen, and vibrate the filter membrane by ultrasonic waves to disperse and enrich the pathogen into the nucleic acid-free buffer.

Preferably, in the step 3), the dry powder reaction system includes the reverse transcription-loop mediated reaction system of H1N1 influenza virus, enterovirus type 71 reverse transcription-loop mediated reaction system, Coxsackie virus A group 16 Type reverse transcription-loop-mediated reaction system.

Preferably, in the step 4), the method for collecting the turbidity of the precipitate produced by the reaction between the enriched solution and the reaction system without adding the dye is to set the LED emission end and the LED light receiving end at the two ends of the micro-reaction chamber, respectively, Collect the electrical signals of the LED transmitting end and the LED light receiving end respectively to obtain the emitted light intensity value I _LED and the received light intensity value I _PD , and calculate the precipitation turbidity:

Turbidity=In( _IPD / _ILED ).

Preferably, the pathogen target curve in the step 5) includes H1N1 type standard curve: y=0.2394x-6.3545, enterovirus type 71 standard curve: y=0.1983x-3.9727, Coxsackie virus group A type 16 standard curve : y=0.1994x-5.9857.

Polymerase chain reaction (PCR) is a high-efficiency in vitro amplification technology of DNA strands. Under specific conditions in vitro, a single DNA strand can be amplified to several 10 ⁷ copies within a few hours, greatly increasing the initial content and specificity of detection. sex. Loop-mediated isothermal amplification (LAMP) in PCR amplification is a technique for amplifying single-stranded RNA, which can amplify a single DNA strand to several 10 ^7-10 copies within a few hours, while the virus in the air is large Most of them are single-stranded RNA, such as influenza virus A H1N1, enterovirus type 71 that causes hand, foot and mouth disease, and Coxsackie virus group A type 16, so LAMP is an existing method to efficiently amplify pathogens in the air in a short time. one of the most effective methods.

The present invention has the following advantages:

1. Fast: From enrichment of viral pathogens to LAMP reaction amplification to the final result warning, the whole process only takes about 100 minutes (all three examples are 100 minutes), while the conventional method takes more than 24 hours;

2. Small: the enrichment bin is as small as cubic centimeters, the thickness of the microfluidic chip board is at the level of millimeters, and the diameter is at the level of centimeters. With other spare parts, the small size makes portability possible;

3. Integrated and intensive operation: the automatic liquid injection device is used, and the enrichment bin, microfluidic chip board and corresponding pipeline are all replacement parts, which avoids pollution and does not require manual operation. It only needs to operate a few buttons. can be completed;

4. High efficiency and high precision: LAMP amplification technology requires low RNA concentration, so the initial concentration of viral pathogens to be enriched is less than that of ordinary PCR amplification, so the enrichment bin is small and the enrichment time is short, making high efficiency possible;

5. The warning effect is clear at a glance: set the qualitative observation window and the quantitative result display screen;

6. Easy to operate: It can automatically monitor in real time, only need to replace the relevant accessories regularly, and operate the relevant interface, which can also be used by ordinary people, not only limited to scientific researchers.

The invention evolves the scientific research work that originally required manual multi-department cooperation into intensive, integrated and miniaturized equipment, so as to meet the actual market demand for shorter, faster, qualitative and quantitative simple operations, and can convert experimental data. Through the Internet reflected as ordinary public information to meet the actual needs of the general public.

Description of drawings

FIG. 1 is a schematic structural diagram of a rapid detection and early warning system for environmental pathogens based on a microfluidic chip of the present invention.

Figure 2 is a schematic diagram of the structure of the air intake device.

Figure 3 is a schematic diagram of the overall structure of the enrichment bin.

Figure 4 is a schematic diagram of the internal structure of the enrichment bin.

Figure 5 is a top view and a side view of the enrichment bin.

Fig. 6 is the internal membrane structure diagram of the enrichment chamber.

FIG. 7 is a schematic structural diagram of a liquid injection device.

FIG. 8 is a schematic structural diagram of a microfluidic chip board.

FIG. 9 is a side view, a top view and a partial enlarged view of the microfluidic chip board.

FIG. 10 is a schematic view of the structure of the observation window.

FIG. 11 is a color-concentration correspondence table in the contrast color chart in the observation window.

In the figure: air inlet device 1, enrichment bin 2, air inlet A, air outlet B, liquid outlet C, liquid inlet D, microporous filter membrane 2.1, upper membrane rear mesh groove liner 2.2, lower membrane front Collection net tank 2.3, air pump 3, ultrasonic vibration plate 4, first quantitative injection device 5A, second quantitative injection device 5B, base 5.1, push rod 5.2, cavity 5.3, servo motor 5.4, microfluidic chip Plate 6, injection hole 6.1, sample flow channel 6.2, micro-reaction chamber 6.3, LED emission end 6.4, LED light receiving end 6.5, temperature measurement plate 7, temperature control plate 8, observation window 9, observation port 9.1, color comparison Card 9.2, first solenoid valve 10, second solenoid valve 11, third solenoid valve 12, fourth solenoid valve 13, fourth solenoid valve 14.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

The microfluidic chip-based rapid detection and early warning system for environmental pathogens proposed by the present invention includes an enrichment component, a microreaction component, a communication component and an early warning component.

Enrichment component: used to suck the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured in the nucleic acid-free buffer. A concentrated solution is formed.

As shown in FIG. 1 , the enrichment assembly includes an air pump 3. The air pump 3 is connected to the air outlet B located at the top of the enrichment bin 2 through the second solenoid valve 11 and a pipeline. The bottom of the enrichment bin 2 is provided with an ultrasonic vibration plate. 4. The side of the enrichment bin 2 is provided with an air inlet A, a liquid inlet D and a liquid outlet C. As shown in Figures 2 and 3, the inner cavity of the enrichment bin 2 is provided with a microporous filter membrane 2.1. The microporous membrane 2.1 is a 0.01um polytetrafluoroethylene membrane (PTFE) with a diameter of 1cm or a polyvinylidene fluoride membrane (PVDF) with a molecular weight cut-off of 100,000, which can retain viral pathogens, but bacterial pathogens can pass through without being retained. The microporous filter membrane 2.1 is sandwiched in the middle and fixed by the upper and lower honeycomb mesh grooves. The diameter of the upper film rear mesh groove liner 2.2 and the lower film front collecting mesh groove 2.3 is 1 cm, and an extremely thin sealing ring is sealed and fixed between the three. The upper membrane rear mesh slot liner 2.2 and the lower membrane front collecting mesh slot 2.3 take the center as a dot, and make circles with radii of 0.1, 0.2, 0.3, and 0.4cm respectively, and the center and the boundary of each circle are evenly distributed on the diameter of the circle. The pores are 0.02cm, so that air and liquid can pass through evenly and balance the pressure on the microporous membrane.

As shown in Figures 1 and 2, the air inlet A is connected to the air inlet device 1 through the first solenoid valve 10, and the liquid inlet D is connected to the first quantitative liquid injection device 5A through the third solenoid valve 12 and the fourth solenoid valve 13 . The second quantitative liquid injection device 5B is connected, and the liquid outlet C is connected to the injection hole 6.1 of the microfluidic chip board 6 in the microreaction assembly through the fourth solenoid valve 14 .

As shown in Figures 1 and 7, the first quantitative liquid injection device 5A and the second quantitative liquid injection device 5B have the same structure, similar to the structure of a syringe, and are provided with a base 5.1, a push rod 5.2 and a cavity 5.3, and the push rod 5.2 And the cavity 5.3 is arranged on the base 5.1, the rear end of the cavity 5.3 is communicated with the push rod 5.2, the front end is communicated with the liquid inlet D through a solenoid valve, the servo motor 5.4 is connected with the rear end of the push rod 5.2, and the push rod 5.2 is connected to the servo Driven by the motor, it moves in the cavity 5.3. The cavity 5.3 of the first quantitative liquid injection device 5A is provided with a nucleic acid-free buffer. When the viral pathogen is trapped by the membrane in the enrichment chamber, the nucleic acid-free buffer in the first quantitative liquid injection device 5A is pushed through the conduit by the push rod 5.1 and flows into the enrichment chamber 2 . When the enrichment solution is to be introduced into the microfluidic chip plate 6, the pusher 5.1 in the second quantitative liquid injection device 5B is used to drive the enrichment solution into the microfluidic chip. The composition of the nucleic acid-free buffer is: 200 mM Tris-HCl (pH 8.8 at 25 degrees Celsius),

100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCL, 20 mM MgSO ₄ , 1% (v/v) Tritonx-100.

Micro-reaction component: It is used to pump the enrichment solution to the microfluidic chip plate for LAMP amplification, that is, to amplify the nucleic acid fragments of specific pathogens, so that the enrichment solution is respectively mixed with the reaction system reagents with dyes and the reagents without dyes. The reaction system reagents are reacted, and the qualitative results of the reaction between the enrichment solution and the dye-added reaction system reagents are displayed through a transparent window.

As shown in FIG. 1 , FIG. 9 , and FIG. 10 , the microfluidic chip assembly includes an observation window 9 , a microfluidic chip board 6 , a temperature measurement board 7 and a temperature control board 8 arranged in order from top to bottom. The microfluidic chip board 6 is made of polydimethylsiloxane polymer (pdms) with good optical transparency and elasticity, and is in the shape of a micro-disc with a thickness not exceeding 50um and a diameter not exceeding 6cm. As shown in FIG. 8 , the microfluidic chip board 6 includes a sample injection hole 6.1 arranged in the center. The bottom of the sample injection hole 6.1 is communicated with the output end of the enrichment component, and 12 are arranged along the periphery of the sample injection hole 6.1. Sample flow channel 6.2, 12 sample flow channels with a width of 50um, a depth of 20um and a length of 150um. The end of each sample flow channel 6.2 is provided with a micro-reaction chamber 6.3 with a volume of 30 uL. An observation port 9.1 is provided at a position corresponding to the observation window 9 and the micro-reaction chamber 6.3, and a comparison color chart 9.2 corresponding to the pathogen is provided below the observation port 9.1.

As shown in FIG. 8 , the micro-reaction chambers 6.3 on the microfluidic chip board 6 are respectively arranged in two areas of qualitative detection and quantitative detection. The micro-reaction chamber 6.3 located in the qualitative detection area is opposite to the observation port 9.1. The inner cavity of 6.3 is provided with a dry powdery reagent with dye added, and the two ends of the micro-reaction chamber 6.3 located in the quantitative detection area are respectively provided with an LED emitting end 6.4 and an LED light receiving end 6.5. The LED transmitting end 6.4 is an LED light source (including a self-measurement light intensity component), and the LED light receiving end 6.5 is an LDR photoresistor (including a received light intensity measuring component). The LED emitting end 6.4 and the LED light receiving end 6.5 are electrically connected with the communication component, and the inner cavity of the micro-reaction chamber 6.3 is provided with a dry powdery reagent without adding dye.

Dry powder reagents have been pre-installed in the reaction microchamber 6.3: the qualitative area is 25mmol/L dNTP, 20umol/L primer FIP, 20umol/L primer BIP, 5umol/L primer F3, 5umol/L primer B3, Bst DNA Polymerase (8U/uL), AMV Reverse Transcriptase (8U/uL), Calcein dye 2uL; quantitative area is the reaction time, 25mmol/L dNTP, 20umol/L primer FIP, 20umol/L primer BIP, 5umol/L primer F3 , 5umol/L primer B3, Bst DNA polymerase (8U/uL), AMV reverse transcriptase (8U/uL). After the reaction buffer containing the enriched sample enters the micro-reaction chamber 6.3, the dry powdered reagents are dissolved and reacted. The dry powder reagent is to freeze the moisture in the reagent in advance by the vacuum freeze-drying method of the freeze dryer, and then sublime the frozen moisture in the reagent in a vacuum aseptic environment to obtain a dry powder reagent formed by freeze drying. ; 8 kinds of reagents can be immobilized on the microfluidic chip plate 6 .

The enrichment bin 2 , the first quantitative liquid injection device 5A and the microfluidic chip board 6 are replacement devices, which need to be replaced together with the connecting pipeline after one use.

After the enriched liquid enters each micro-reaction chamber 6.3, the dry powdered reagent in the reaction chamber is dissolved, and the microfluidic chip board 6 reacts through the heating and temperature control device, and the reactions in the qualitative area and the quantitative area start at the same time. In the qualitative area, finally the dye produces a color in the micro-reaction chamber, which is compared with the color chart in the display window to confirm whether the virus is present, and if there is a qualitative judgment of the concentration range. As shown in Figure 10, after the micro-reaction is completed, regarding the qualitative reaction, observe from the observation window 9.1. If there is no corresponding virus in the air, the micro-reaction chamber 6.3 will appear orange. If there is a corresponding virus in the air, the micro-reaction chamber 6.3 is yellow-green, and according to the degree of yellow-green and the comparison color card 9.2 in the colorimetric window 9, the range of virus content in the air is obtained. As shown in Figure 11, the yellowish green in the table shows that the virus concentration is below 10copies/ ^m3 , and the yellowish green in the table shows that the virus concentration is 10-100copies/m between the middle yellowish green and light yellowish green. ^3. Yellow-green in the middle yellow-green and dark yellow-green in the table shows that the virus concentration is 100-500copies/m ³ , and the yellow-green in the table with dark yellow-green or darker indicates that the virus concentration is more than 500 copies/m ³ . In the quantitative area, no dye is added, and a white precipitate of pyrophosphate produced by the reaction can be observed. The photoelectric element is used to detect the turbidity of the precipitate produced by the reaction, and then the analog-to-digital converter is used to convert the optical signal into an electrical signal, and then pass the remote The server or MCU and the application program calculate the virus content to quantify, and the test results can be directly observed through the remote terminal, which is convenient for ordinary people to directly observe.

Communication component: used to receive the electrical signal collected by the optoelectronic equipment, wirelessly transmit the electrical signal to the remote server, receive the quantitative detection result and early warning instruction sent by the remote server, and send it to the display device and early warning component; the remote server calculates according to the electrical signal Precipitation turbidity value and virus concentration, according to the virus concentration and pathogen target curve output reaction quantitative detection results and early warning instructions; the process of calculating the sediment turbidity value and virus concentration can also be completed locally.

The communication component includes A/D converter, MCU and wireless communication module. The A/D converter is used to receive electrical signals from the LED transmitter 6.4 and the LED light receiver 6.5. The MCU is used to wirelessly transmit the electrical signals to the remote through the wireless communication module. The server receives the quantitative reaction detection results and early warning instructions sent by the remote server through the wireless communication module, and sends the quantitative reaction detection results and early warning instructions to the display device and the early warning component. The MCU can also calculate the sediment turbidity value and the virus concentration locally and output the quantitative detection result of the reaction according to the virus concentration and pathogen target curve.

Quantitative judgment: The turbidity of the micro-reaction product pyrophosphate precipitation causes different light intensities, and is then detected by a photosensitive device to indirectly monitor and analyze the amplification. The essence is to monitor the entire process of viral RNA fragment amplification. The time of turbidity formation has a certain functional relationship with the formation of DNA concentration. When LAMP is amplified, it is incubated under isothermal conditions, and the time of turbidity emergence is measured by measuring the time of turbidity. to indirectly quantitatively determine the concentration. As shown in Figures 8 and 9, the light (650 nm) emitted by the LED emitting end 6.4 passes through the micro-reaction chamber 6.3 and illuminates the LED light-receiving end 6.5, and the microfluidic chip board 6 is heated by the temperature control board 8 to each stage of the LAMP reaction Temperature, the temperature of the temperature control panel 8 is controlled by the temperature controller. Time monitoring is monitored by the system. After the digital-to-analog converter (A/D) converts the signal, it receives the electrical signal converted by the luminous intensity (ILED) of the LED transmitting end 6.4 and the light intensity received by the LED light receiving end 6.5 (IPD), and the single-chip microcomputer (MCU) collects the signal, and then Wireless transmission to the server (SERVER), the server background calculation, and finally transmitted to the wireless terminal equipment (RTU).

The server is responsible for processing the data collected from the single-chip microcomputer, forming real-time graphs, and obtaining information such as the content of the target virus through data processing and graph analysis. When working, the server is put into operation, and the system enters the established program until the end of the micro-reaction. At the same time, the system collects the turbidity signal in real time. The server forms a real-time graph according to the collected signal, and performs data processing and graph analysis to obtain the virus to be tested. content.

The server and the single-chip processor are connected by wireless signals, and the control unit is respectively connected to the temperature sensor, the ambient temperature sensor, and the photoelectric system; the temperature sensor is located at the bottom of the microfluidic chip board, and the ambient temperature sensor is located inside the entire system. The photoelectric system has been described in detail. Time detection sensors are located on key components.

The application software is installed on the remote terminal and can run under Windows, Android, and iOS systems to realize experimental setup, data management and other functions. The application software will monitor the temperature control system, optoelectronic system, etc.

Early warning component: used to issue sound and/or light alarm signals according to the early warning instructions sent by the communication component.

The whole system is a linkage operation system. In the startup state, the third solenoid valve 13 and the fourth solenoid valve 14 corresponding to the liquid inlet D and the liquid outlet C are closed, and the first solenoid valve corresponding to the air inlet A and the air outlet B is closed. 10. The second solenoid valve 11 is opened, the pathogens in the air begin to enrich, and the enrichment is completed, the first solenoid valve 10 and the second solenoid valve 11 are closed, the third solenoid valves 12 and 13 (5A passage) are opened, and the first solenoid valve 10 and the second solenoid valve 11 are closed. The push rod 5.1 of the volume injection device 5A injects the nucleic acid-free buffer in the cavity into the liquid inlet D, starts the ultrasonic vibration plate 4, then closes the 5A passage of the third solenoid valve 12, and opens the 5B passage of the third solenoid valve 12. The push rod 5.2 of the two-quantity liquid injection device 5B pushes the enrichment solution into the micro-reaction assembly automatically through the liquid inlet D. After the sample is added, the micro-reaction assembly monitors the reaction through the temperature control device and the temperature sensitive device. a Qualitative reaction: Finally, the dye produces yellow-green in the micro-reaction chamber, and it is compared with the colorimetric card in the display window to confirm whether the virus exists, and if there is a qualitative judgment of the concentration range; b Quantitative reaction: in the microfluidic chip board 6 The photoelectric device detects the turbidity of the precipitate produced by the reaction, and then calculates the virus content through the remote server and application program to quantify. There is no need for operators to do other operations or professional calculations to determine the content, and the calculation results are directly operated by the programmed system, which ensures high precision and integration.

The system can also be provided with a remote mobile terminal, and the remote mobile terminal can receive the quantitative detection results of the reaction by connecting to the remote server. In this way, ordinary people can directly query the test results through remote terminals, such as mobile phones and tablet computers, to understand the environmental virus situation in the monitored area.

The present invention also provides a method for rapid detection and early warning of pathogens in the environment based on microfluidic chip detection, comprising the following steps:

1) Suction the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured in the nucleic acid-free buffer to form enrichment. liquid. The sampling time of 30min, the flow rate of the air pump 3 is about 5L/min, and the virus pathogen in 150L of air in the space to be detected is collected in 30min. After the collection is completed, the enrichment bin 2 uses the first quantitative liquid injection device 5A to introduce a nucleic acid-free buffer, then starts the ultrasonic micro-vibrating disk 4, and runs for 10 minutes at a frequency of 50KHZ to disperse and enrich the viral pathogens on the filter membrane to 1mL reaction buffer. In the liquid, turn off the ultrasonic micro-vibration plate 4. The composition of the nucleic acid-free buffer was 200 mM Tris-HCL (pH 8.8 at 25 degrees Celsius), 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCL, 20 mM MgSO ₄ , 1% (v/v) Tritonx-100.

2) After the set collection time is reached, the enrichment process is completed, and the enrichment solution is injected into the microfluidic chip plate for LAMP amplification, that is, amplification of specific pathogen nucleic acid fragments.

3) After the set amplification time is completed and the amplification process is completed, the amplified enrichment solution is introduced into the micro-reaction chamber to react with the pre-installed dry powder reaction system reagents, and the temperature of the reaction chamber is controlled to 65°C and the reaction time is controlled. 50min, the micro-reaction chamber is divided into two areas: qualitative detection and quantitative detection. The dry powder reaction system reagent in the qualitative detection area is added with dye, and the dry powder reaction system reagent for quantitative detection is not added with dye; dry powder Reaction system reagents include H1N1 influenza virus reverse transcription-loop mediated reaction system reagents, enterovirus type 71 reverse transcription-loop mediated reaction system reagents, Coxsackie virus group A type 16 reverse transcription-loop mediated reaction system reagents reagents.

LAMP amplification reaction system

System Details (Concentration) volume 25mmol/L dNTP 2uL 20umol/L primer FIP 1.5uL 20umol/L primer BIP 1.5uL 5umol/L primer F3 1.5uL 5umol/L primer B3 1.5uL Bst DNA polymerase (8U/uL) 2uL AMV reverse transcriptase (8U/uL) 2uL enriched sample (with reaction buffer) 13uL 25uL system

Primer design for LAMP reaction:

flu virus:

Reverse transcription-loop-mediated isothermal amplification method (RT-LAMP) isothermal amplification primers for H1N1 influenza virus:

F3:AAGACAAGTTCATGGCCCAATC;

B3:CGGCTGCATATCCTGACCC;

FIP: CCGGCTTGAACTTCTTCTTGCTGCTGTAGGGGCATTCACCATCCATCT;

BIP: GTGCTATAAACACCAGCCTCCCAGGCCAGTCTCAATTTTGTGCTT.

HFMD:

Enterovirus 71 (English name: Human enterovirus 71), referred to as EV71, is one of the main pathogens that cause hand, foot and mouth disease in infants and young children. Reverse transcription-loop-mediated isothermal amplification primers

F3:GCGCAAATGCGTAGAAAGG

B3:AGGGTCTGACAGCTTGACAA;

FIP: CAACTTCCCCGGTGGGTGTGTTTTCCTACATGCGCTTTTGATGCA;

BIP: TATGTTTGTGCCACCTGGAGCCTTTTGGGGTTAGTGGCGGTTTG.

Coxsackievirus A group 16 (English name coxsackievirus A16), referred to as CA16, is one of the main pathogens causing hand, foot and mouth disease in infants and young children. Reverse transcription-loop-mediated isothermal amplification primers:

F3:AGGAGACAGCCATTGGGAA;

B3:ACTGCAGTAATTGGGGGACT;

FIP: GTTCTGTGTACCCGTGGTGGGTTTCTTTAGCCGTGCTGGTT;

BIP:TGCTCAATTACGGCGCAAATGCGCTTGGCTACGACAAATGTG.

4) Determine the nature of the pathogen by comparing the color card with the color of the sediment in the micro-reaction chamber in the qualitative detection area. After the micro-reaction is completed, if there is no corresponding virus in the air, the micro-reaction chamber will appear orange. If there is a corresponding virus in the air Viruses, the micro-reaction chamber appears yellow-green. According to the comparison of the degree of yellow-green with the color chart in the colorimetric window, the range of virus content in the air can be obtained.

Collect the precipitation turbidity generated by the reaction between the enrichment solution and the reaction system without dye added by optoelectronic equipment: set the LED emission end 6.4 and the LED light receiving end 6.5 at both ends of the micro-reaction chamber respectively, and collect the LED emission end 6.4 and LED light respectively. The electrical signal at the receiving end 6.5 obtains the emitted light intensity value I _LED , the received light intensity value I _PD , and calculates the precipitation turbidity:

Turbidity=In( _IPD / _ILED ).

5) Calculate the precipitation turbidity value and the virus concentration according to the precipitation turbidity generated by the reaction of the enrichment solution and the reaction system without adding the dye, output the quantitative detection result of the reaction according to the virus concentration and the pathogen target curve, and send out when the detection result exceeds the preset value. Warning order.

Concentration calculation: Use the RNA templates of various virus controls to establish the quantitative relationship between the turbidity peak time and concentration. It is found that their peak peak time has a stable linear relationship with the concentration value at the base of 10, which can be used as a standard curve. Realize quantitative detection of samples.

H1N1 standard curve drawing: one RNA sample with concentrations of 10 ⁰ ng/uL, 10 ¹ ng/uL, 10 ² ng/uL, 10 ³ ng/uL, 10 ⁴ ng/uL, and 10 ⁵ ng/uL. Use the above system to capture the peak time of turbidity, and make a negative logarithmic relationship table between the peak time of turbidity and the concentration when the H1N1 standard curve is drawn, as shown in Table 1, and make a standard curve with the above concentration to obtain Equation: y=0.2394x-6.3545. From the standard curve, the correlation coefficient is R2=0.9899, showing a good correlation linear relationship. During the test, according to the measured peak time of the target enriched virus reaction, the enriched virus concentration S (value) can be calculated according to the standard curve, and the target virus concentration in the measured area can be calculated as 20/3*S mg /M ³ .

Table 1. Negative logarithmic relationship between turbidity peak time and concentration when drawing H1N1 standard curve

time (min) 26.5 31.2 33.8 40.1 43.6 46.75 -log of concentration 0 1 2 3 4 5

Enterovirus 71 standard curve drawing: RNA samples with concentrations of 10 ⁰ ng/uL, 10 ¹ ng/uL, 10 ² ng/uL, 10 ³ ng/uL, 10 ⁴ ng/uL, and 10 ⁵ ng/uL each , you can use the above system to capture the peak time of turbidity, and make a negative logarithmic relationship table between the peak time of turbidity and the concentration when the enterovirus type 71 standard curve is drawn, as shown in Table 2. A standard curve was obtained to obtain the equation: y=0.1983x-3.9727. From the standard curve, the correlation coefficient is R2=0.9861, showing a good correlation linear relationship. During the test, according to the measured peak time of the target enriched virus reaction, the enriched virus concentration S (value) can be calculated according to the standard curve, and the target virus concentration in the measured area can be calculated to be 20/3*S mg /M ³ .

Table 2

Negative logarithmic relationship table of turbidity peak time and concentration when drawing standard curve of enterovirus type 71

time (min) 20.5 24.6 29.3 36.1 41.6 43.75 -log of concentration 0 1 2 3 4 5

Coxsackie virus group A 16 standard curve drawing: concentrations of 10 ⁰ ng/uL, 10 ¹ ng/uL, 10 ² ng/uL, 10 ³ ng/uL, 10 ⁴ ng/uL, 10 ⁵ ng/uL One RNA sample each, the peak time of turbidity can be captured by the above system, and the negative logarithmic relationship between the peak time of turbidity and the concentration when the standard curve of Coxsackie virus A group 16 is drawn, as shown in Table 3 shown, and made a standard curve with the above concentrations to obtain the equation: y=0.1994x-5.9857. From the standard curve, the correlation coefficient is R2=0.9909, showing a good correlation linear relationship. During the test, according to the measured peak time of the target enriched virus reaction, the enriched virus concentration S (value) can be calculated according to the standard curve, and the target virus concentration in the measured area can be calculated to be 20/3*S mg /M3.

table 3

Negative logarithmic relationship between turbidity peak time and concentration when the standard curve of Coxsackie virus group A type 16 is drawn

time (min) 30.3 35.7 39.3 43.9 51.3 54.8 -log of concentration 0 1 2 3 4 5

Example 1:

The conference room of a company is 30m ² . On September 20, 2018, there were 10 people in the conference room.

Place the device in the center of the classroom, and the whole process takes about 100 minutes.

a.30min sampling time, about 150L of indoor air will flow through the sampling device through the air inlet device 1 and the air pump 3, and the membrane in the enrichment device will trap the virus in the air;

b. The ultrasonic vibration plate 4 is shaken for 10 minutes, and the virus on the filter membrane is evenly dispersed into 1ml of reaction buffer, and 1ml of reaction buffer is injected into the enrichment chamber 2 by the quantitative liquid injection device;

c. Inject 60uL of the enrichment solution into the central hole of the microfluidic chip plate 6, then evenly inject 13uL into each microreaction chamber 6.3, wait for 1min, the lyophilized reagent in the microreaction chamber 6.3 is completely dissolved in the buffer solution medium, about 1min;

d. Start the LAMP amplification reaction on the microfluidic chip plate 6 at 65° C. for 50 min.

e. During the reaction, A: Qualitative detection of all the corresponding holes of the target virus is orange, basically confirming that there is no influenza virus, hand, foot and mouth virus. B: Quantitative detection, no turbidity peaks were observed for all target viruses in the remote terminal software program, indicating that the reaction did not produce pyrophosphate precipitation, and it was determined that there was no influenza virus or hand-foot-mouth virus.

Example 2:

The second grade classroom of a primary school is 30m ² . On September 20, 2018, 3 students in the classroom had cough symptoms.

Place the device in the center of the classroom, and the whole process takes about 100 minutes.

a. 30min sampling time, about 150L of indoor air will flow through the sampling device through the air intake device 1 and the air pump 3, and the membrane in the enrichment device will trap the virus in the air

b. The ultrasonic vibration plate 4 was shaken for 10 minutes, and the virus on the filter membrane was evenly dispersed into 1ml of reaction buffer, and 1ml of reaction buffer was injected into the enrichment chamber by the quantitative injection device

c. Inject 60uL of the enrichment solution into the central hole of the microfluidic chip plate 6, then evenly inject 13uL into each microreaction chamber 6.3, wait for 1min, the lyophilized reagent in the microreaction chamber 6.3 is completely dissolved in the buffer solution middle. about 1min

d. Start the LAMP amplification reaction on the microfluidic chip board at 65°C for 50min.

e. During the reaction process, the corresponding hole for qualitative detection of H1N1 influenza virus is yellow-green, and the virus concentration is within 500copies/m ³ according to the color card, and the corresponding detection holes for other viruses are orange. oral virus. B: Quantitative detection, the turbidity peak curve was observed for influenza virus in the remote terminal software program, and the peak emergence time was 21.7min. The remote application program directly calculated the concentration of influenza virus in the air according to the formula is about 14.2mg/M3, and other target viruses If there is no turbidity outburst curve, there is no other target to detect the virus.

Example 3

A classroom is 30m ² . On September 5, 2018, there were students in the class who asked for leave to go home because of hand, foot and mouth disease. Now the whole class is resting for half a day, and the classroom is ready to be disinfected immediately.

Place the device in the center of the classroom, and the whole process takes about 100 minutes.

a.30min sampling time, about 150L of indoor air will flow through the sampling device through the air inlet device 1 and the air pump 3, and the membrane in the enrichment device will trap the virus in the air;

b. The ultrasonic vibration plate 4 is shaken for 10 minutes, and the virus on the filter membrane is evenly dispersed into 1ml of reaction buffer, and 1ml of reaction buffer is injected into the enrichment chamber 2 by the quantitative liquid injection device;

c. Inject 60uL of the enrichment solution into the central hole of the microfluidic chip plate 6, and then evenly inject 13uL into each microreaction chamber 6.3, wait for 1min, the lyophilized reagent in the microreaction chamber 6.3 is completely dissolved in the buffer in liquid. about 1min;

d. Start the LAMP amplification reaction on the microfluidic chip board at 65°C for 50min.

e. During the reaction, a yellow-green color was observed in the corresponding hole of the Coxsackie virus group A type 16 in qualitative detection. Compared with the color card, the virus concentration was above 500 copies/m ³ , and the corresponding detection holes of other viruses were orange-yellow. Hand, foot and mouth virus, no influenza virus. B: Quantitative detection, the turbidity peak curve of Coxsackie virus A group 16 was observed in the remote terminal software program, and the peak time was 31.2min. The remote application program directly calculated the concentration of influenza virus in the air according to the formula is about 11.5mg /M3, other target viruses have no turbidity peak curve, and no other target viruses can be detected.

Those skilled in the art should understand that the specific embodiments described herein are only used to explain the patent of the present invention, and are not used to limit the patent of the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the patent of the present invention shall be included in the protection scope of the patent of the present invention.

Claims (17)

1. a microfluidic chip-based rapid detection and early warning system for environmental pathogens, characterized in that: the system comprises enrichment components, microreaction components, communication components and early warning components; The enrichment component: used to suck the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured to the nucleic acid-free. The enrichment solution is formed in the buffer; The micro-reaction component is used to pump the enrichment solution to the microfluidic chip plate for LAMP amplification, that is, to amplify the nucleic acid fragments of specific pathogens, so that the enrichment solution is respectively mixed with the reaction system reagents added with dyes, and the non-added dyes. The dye's reaction system reagents react, and the qualitative results of the reaction between the enrichment solution and the dye-added reaction system reagents are displayed through a transparent window, and the precipitation turbidity generated by the reaction between the enrichment solution and the dye-free reaction system reagents is collected by photoelectric equipment; The communication component: used to receive the electrical signal collected by the optoelectronic equipment, wirelessly transmit the electrical signal to the remote server, receive the quantitative detection result of the reaction and the early warning instruction sent by the remote server, and send it to the display device and the early warning component; the remote server Calculate the precipitation turbidity value and virus concentration according to the electrical signal, and output the quantitative detection results and early warning instructions according to the virus concentration and pathogen target curve; The pre-warning component: used to issue a sound and/or light alarm signal according to the pre-warning instruction sent by the communication component. 2. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 1, wherein the enrichment component comprises an air pump (3), and the air pump (3) passes through a solenoid valve and a The pipeline is connected to the air outlet (B) located at the top of the enrichment bin (2), the bottom of the enrichment bin (2) is provided with an ultrasonic vibration plate (4), and the side of the enrichment bin (2) is provided with Air inlet (A), liquid inlet (D) and liquid outlet (C), a microporous filter membrane (2.1) is arranged in the inner cavity of the enrichment bin (2), and the microporous filter membrane ( 2.1) It is covered and fixed by the upper film rear mesh groove liner (2.2) and the lower film front collecting mesh groove (2.3). The air inlet (A) is connected to the air inlet device (1) through a solenoid valve. The liquid port (D) is connected to the first quantitative liquid injection device (5A) and the second quantitative liquid injection device (5B) through a solenoid valve, and the liquid outlet (C) is connected to the microfluidic chip assembly. 3. The rapid detection and early warning system for environmental pathogens based on a microfluidic chip according to claim 1, wherein the microreaction assembly comprises an observation window (9), a microfluidic chip, which are arranged in sequence from top to bottom. A plate (6), a temperature measurement plate (7) and a temperature control plate (8), the microfluidic chip plate (6) comprises a sample injection hole (6.1) arranged in the center, and the sample injection hole (6.1) has a The bottom is communicated with the output end of the enrichment component, a plurality of sample flow channels (6.2) are arranged along the periphery of the sample injection hole (6.1), and a micro-reaction chamber is arranged at the end of each sample flow channel (6.2). (6.3), an observation port (9.1) is provided at the corresponding position of the observation window (9) and the micro-reaction chamber (6.3), and a color comparison card (9.2) corresponding to the pathogen is provided below the observation port (9.1). . 4. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 2, characterized in that: the structure of the first quantitative liquid injection device (5A) and the second quantitative liquid injection device (5B) The same, all are provided with a base (5.1), a push rod (5.2) and a cavity (5.3), the push rod (5.2) and the cavity (5.3) are arranged on the base (5.1), one end of the cavity (5.3) It is communicated with the push rod (5.2), and the other end is communicated with the liquid inlet (D) through a solenoid valve. The cavity (5.3) of the first quantitative liquid injection device (5A) is provided with a nucleic acid-free buffer. There is no liquid in the cavity (5.3) of the second quantitative liquid injection device (5B), and sterile and virus-free air is stored. 5. The rapid detection and early warning system for environmental pathogens based on a microfluidic chip according to claim 3, characterized in that: the microreaction chambers (6.3) on the microfluidic chip board (6) are respectively arranged for qualitative detection In the two areas of quantitative detection and quantitative detection, the micro-reaction chamber (6.3) located in the qualitative detection area is opposite to the observation port (9.1). Two ends of the micro-reaction chamber (6.3) in the quantitative detection area are respectively provided with an LED emitting end (6.4) and an LED light receiving end (6.5), and the LED emitting end (6.4) and the LED light receiving end (6.5) communicate with The components are electrically connected, and the inner cavity of the micro-reaction chamber (6.3) is provided with a dry powder reagent without dye added. 6. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 5, wherein the communication component comprises an A/D converter, an MCU and a wireless communication module, and the A/D converter The MCU is used to receive electrical signals from the LED transmitter (6.4) and the LED light receiver (6.5), the MCU is used to wirelessly transmit the electrical signals to the remote server through the wireless communication module, and receive the response sent by the remote server through the wireless communication module Quantitative detection results and early warning instructions, and send the response quantitative detection results and early warning instructions to the display device and early warning components. 7. The rapid detection and early warning system for environmental pathogens based on a microfluidic chip according to claim 2, wherein the microporous filter membrane (2.1) is a filter membrane with a pore diameter≤0.01um, such as polytetrafluoroethylene Membrane PTFE or polyvinylidene fluoride membrane PVDF or polyvinylidene fluoride membrane VDF with a molecular weight cut-off of 100,000. 8. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 2, characterized in that: the upper film rear mesh groove liner (2.2) and the lower film front collection mesh groove (2.3) The diameter is a cm, a ≥ 1, the center is the dot, and the radius is 0.1a, 0.2a, 0.3a, 0.4a cm respectively. The radius is a circle, and the center and the boundary of each circle are uniformly distributed with a diameter of 0.02a cm holes. 9 . The rapid detection and early warning system for environmental pathogens based on a microfluidic chip according to claim 1 , wherein the material of the microfluidic chip board (6) is made of polydimethylsiloxane polymer PDMS. 10 . to make. 10. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 1, characterized in that: the enrichment bin (2), the first quantitative liquid injection device (5A) and the microfluidic control The chip board (6) and the corresponding pipeline are replacement devices after use. 11. The microfluidic chip-based rapid detection and early warning system for environmental pathogens according to claim 1, wherein the system further comprises a remote mobile terminal, and the remote mobile terminal receives the quantitative detection result of the reaction by connecting to a remote server . 12. A method for rapid detection and early warning of environmental pathogens based on a microfluidic chip, wherein the method comprises the following steps: 1) Suction the air in the space to be detected, trap the pathogens in the air on the filter membrane through the structural parts, and then dissolve them in the nucleic acid-free buffer, so that the pathogens in the air are captured in the nucleic acid-free buffer to form enrichment. liquid; 2) The enrichment solution is injected into the microfluidic chip plate for LAMP amplification, that is, the amplification of specific pathogen nucleic acid fragments; 3) The amplified enrichment solution is introduced into the micro-reaction chamber to react with the pre-installed dry powder reaction system, and the temperature and reaction time of the reaction chamber are controlled. The micro-reaction chamber is divided into two areas: qualitative detection and quantitative detection. , the dry powder reaction system reagent in the qualitative detection area is added with dye, and the dry powder reaction system reagent in the quantitative detection area is not added with dye; 4) Determine the concentration range of viral pathogens by comparing the color of the reaction solution in the micro-reaction chamber of the qualitative detection area with the color card, and collect the turbidity of the precipitate generated by the reaction between the enrichment solution and the reaction system reagents without adding dyes through the photoelectric device, thereby indirectly Monitoring viral pathogen concentrations; 5) Calculate the precipitation turbidity value and the virus concentration according to the precipitation turbidity generated by the reaction between the enrichment solution and the primer without adding dye, output the quantitative detection result of the reaction according to the virus concentration and the pathogen target curve, and issue an early warning when the detection result exceeds the preset value instruction. 13. The method for rapid detection and early warning of environmental pathogens based on a microfluidic chip according to claim 12, wherein the composition of the nucleic acid-free buffer in the step 1) is 200 mM Tris-HCL (pH 8.8 at 25 degrees Celsius) ), 100 mM (NH ₄ ) ₂ SO ₄ , 100 mM KCL, 20 mM MgSO ₄ , 1% (v/v) Tritonx-100. 14. The method for rapid detection and early warning of environmental pathogens based on a microfluidic chip according to claim 13, wherein the method for dissolving viral pathogens in the nucleic acid-free buffer in the step 1) is to submerge the nucleic acid-free buffer. The filter membrane that retains pathogens is shaken by ultrasonic waves to disperse and enrich the pathogens into a nucleic acid-free buffer. 15. The method for rapid detection and early warning of environmental pathogens based on a microfluidic chip according to claim 13, characterized in that: in the step 3), the dry powder reaction system comprises reverse transcription-loop mediation of H1N1 influenza virus Reaction system reagents, enterovirus type 71 reverse transcription-loop-mediated reaction system reagents, Coxsackie virus group A type 16 reverse transcription-loop-mediated reaction system reagents. 16. The method for rapid detection and early warning of environmental pathogens based on a microfluidic chip according to claim 13, characterized in that: in the step 4), the enrichment solution is collected by optoelectronic equipment and the reaction of the dye-free reaction system reagent is generated. The method of precipitating turbidity is to set the LED emitting end (6.4) and the LED light receiving end (6.5) at both ends of the micro-reaction chamber, respectively, and collect the electricity of the LED emitting end (6.4) and the LED light receiving end (6.5) respectively. The signal obtains the emitted light intensity value I _LED , the received light intensity value I _PD , and calculates the precipitation turbidity: Turbidity=In( _IPD / _ILED ). 17. The method for rapid detection and early warning of environmental pathogens based on a microfluidic chip according to claim 13, wherein the pathogen target curve in the step 5) comprises an H1N1 type standard curve: y=0.2394x-6.3545, intestinal Channel virus type 71 standard curve: y=0.1983x-3.9727, Coxsackie virus group A 16 standard curve: y=0.1994x-5.9857.

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