CN104360060A - Detection method for specific antibodies IgM of mycoplasma pneumonia and influenza viruses based on micro-fluidic chip - Google Patents

Abstract

The invention relates to a detection method for specific antibodies IgM of mycoplasma pneumonia and influenza viruses based on a micro-fluidic chip and application of the detection method. The method is characterized by taking the micro-fluidic chip with a plurality of pipelines as a reaction platform and detecting by using a chemical luminescent signal so as to independently or simultaneously detect the serum specific antibodies IgM of mycoplasma pneumonia and influenza viruses. As the micro-fluidic chip is combined with chemiluminiscence, the detection method integrates the advantages of rapidness, high efficiency and small sample amount of the micro-fluidic chip and high specificity of chemiluminiscence, also has the advantages of simplicity in operation, low cost, rapidness and the like, is a multi-target, real-time and high-sensitivity detection method and can be applied to rapid diagnosis of pneumonia and influenza.

Description

A microfluidic chip-based detection method for Mycoplasma pneumoniae and influenza virus-specific antibody IgM

technical field

The invention relates to the technical field of biological analysis, in particular to a method for detecting Mycoplasma pneumoniae and influenza virus, and in particular to a method for detecting the specific antibody IgM of Mycoplasma pneumoniae and influenza virus based on a microfluidic chip.

Background technique

Mycoplasma pneumoniae (Mycoplasma Pneumonia) is the pathogen of human mycoplasma pneumonia, which is characterized by paroxysmal irritating cough. In addition to the manifestations of the respiratory system, mycoplasma pneumonia can be accompanied by multi-system and multi-organ damage. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes accompanied by bronchial pneumonia, which is called primary atypical pneumonia. It is mainly transmitted by droplets, with an incubation period of 2 to 3 weeks. Some clinical symptoms are mild, or even asymptomatic. If there are, they are just general respiratory symptoms such as headache, sore throat, fever, and cough. The clinical manifestations and chest X-ray examination of mycoplasma pneumonia are not characteristic, and the diagnosis cannot be made based on clinical manifestations and chest X-ray examination alone.

Influenza virus, mainly causing human influenza, is a highly contagious acute respiratory infectious disease. Influenza epidemics are accompanied by increased mortality, and the increased mortality is not only caused by influenza and pneumonia, but also related to the exacerbation of cardiopulmonary disease and other chronic diseases caused by influenza. It is characterized by rapid onset, chills, fever, body temperature rises to a peak within a few hours to 24 hours, and usually has myositis and gastrointestinal symptoms. The epidemic is mainly in winter and spring. In the early stage of the epidemic, it is difficult to diagnose sporadic or mild cases, and laboratory tests are often required for diagnosis.

Immunoglobulin M (IgM) and immunoglobulin G (IgG) are the two most important antibodies in humans or animals. When the body is infected with Mycoplasma pneumoniae or influenza virus, in the body, the specific IgM antibody of Mycoplasma pneumoniae or influenza virus appears first, and then the specific IgG antibody of Mycoplasma pneumoniae or influenza virus appears. Therefore, by detecting the presence or absence of Mycoplasma pneumoniae or influenza virus-specific IgM and IgG antibodies in the body, the immune response state of humans or animals to pathogens can be diagnosed.

At present, the specific IgM of Mycoplasma pneumoniae or influenza virus in samples is mainly detected by enzyme-linked immunosorbent assay kit. The detection products are basically divided into three categories: one is the traditional microplate product, its disadvantage is that the detection process takes a long time (usually several hours), and the amount of reagents and samples used is also large, which cannot meet the needs of a small number of samples. Or the need for timely clinical detection; one type is fluorescent labeling detection slide products, which use less reagents, but the detection time is still long, and most of the results of such products need to be observed and interpreted with the help of expensive fluorescence microscopes , this kind of intuitive judgment requires the rich experience of technicians and is time-consuming, so it is not suitable for rapid detection on the spot; the other type is colloidal gold test strips, which are relatively fast and simple, but their application has certain limitations and cannot be quantified. And it cannot meet the simultaneous detection of multi-indicators and multi-samples.

In addition, although the current products for detecting pathogen serum are relatively mature, the most common products on the market are microwell plates and test strip products, most of which are specific IgM detection products for a certain pathogen serum, and few Products for joint detection of multiple pathogens.

CN201804009U discloses a detection method based on a microfluidic chip pathogen, which adopts a microchannel and a detection chip on the body, and each detection chip is connected to the microchannel, and each chip is combined with a kind of antibody, and various antibodies are different. The disadvantage of this method is that if multiple indicators are detected, multiple detection chips need to be used, which are partitioned, which inevitably increases the experimental steps and makes the operation cumbersome. In addition, the preparation method involved is to coat the antibody to detect the antigen, which cannot be completed The purpose of detecting serum-specific antibodies.

CN102854304A discloses a microfluidic chip with an integrated micro-magnetic field as a reaction container, magnetic balls as a solid phase carrier, and streptavidin-modified quantum dots (SA-QDs) as fluorescent markers. Next, the magnetic ball is captured at a specific part of the chip channel to form a micro-reaction area, and the pathogen is captured through the sandwich immune reaction, and the fluorescent immunoquantitative analysis of the pathogen is realized through the interaction between biotin and streptavidin. Since the detection method involves biotin, there may be some interference due to the biotin contained in the human body sample components. In addition, the cost of reagents and equipment used in the fluorescence quantitative analysis is relatively high.

Therefore, finding a multi-target, real-time, and highly sensitive method for detecting Mycoplasma pneumoniae and influenza virus is an urgent problem to be solved at present.

Contents of the invention

The purpose of the present invention is to provide a detection method for Mycoplasma pneumoniae and influenza virus, in particular to a detection method for specific antibody IgM of Mycoplasma pneumoniae and influenza virus based on a microfluidic chip.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a method for detecting the specific antibody IgM of Mycoplasma pneumoniae and influenza virus based on a microfluidic chip, comprising the following steps:

(1) Fabrication of the detection chip: the chip is attached to the solid-phase reaction substrate to form a closed microfluidic channel, and the specific recombinant protein antigen of Mycoplasma pneumoniae and/or influenza virus is introduced into the microfluidic channel, and the The specific recombinant protein antigen is coated on the substrate; after coating, the coating solution is drawn out, the chip is peeled off, and another chip is pasted, so that the channels on the two chips cross each other, and after sealing it with the substrate , pass the blocking liquid into the micro-flow channel, and draw out the blocking liquid after sealing;

(2) adding the sample to be tested to the microfluidic pipeline sample hole in the detection chip described in step (1), incubating and washing;

(3) adding an enzyme-labeled detection antibody to the microfluidic pipeline described in step (2), and washing;

(4) Adding chemiluminescence liquid into the microfluidic pipeline described in step (3), and detecting by a chemiluminescence detector.

The optimized specific recombinant protein antigens of pathogens such as mycoplasma pneumoniae or influenza virus adopted in the present invention can avoid non-specific reactions, can specifically recognize pulmonary branch or influenza IgM antibodies in serum, and react with them to convert serum Medium-specific IgM antibodies remain on the solid substrate, and the rest of the serum components are washed away.

The chip described in the step (1) of the present invention is a chip with parallel microfluidic pipelines that is formed by manual overmolding or injection molding.

Preferably, the chip has 7 parallel microfluidic channels; the length of the microfluidic channels is 3-5cm, the diameter of the microfluidic channels is 500-600 μm, the height is 500-600 μm, and the distance between the microfluidic channels is 1 -3mm.

In the present invention, each microfluidic channel only needs to pass 20 μL or even less detection sample to meet the detection requirements.

The solid phase reaction substrate in the step (1) of the present invention is polystyrene, polydimethylsiloxane or polymethyl methacrylate, preferably polystyrene.

The pipes on the two chips described in step (1) of the present invention are perpendicular to each other, and the intersection angle is 90°.

The tested sample in the step (2) of the present invention is serum.

The invention uses serum samples for detection, and can quickly and accurately diagnose whether human or animal organisms have recent mycoplasma pneumoniae or influenza virus infection.

The incubation time in step (2) of the present invention is 10-20 min, preferably 15 min.

The enzyme-labeled detection antibody in step (3) of the present invention is an optimized HRP-labeled goat anti-human IgM antibody.

The chemiluminescence liquid described in the step (4) of the present invention is a luminol chemiluminescence substrate, and the substrate can be catalyzed by HRP enzyme.

The washing liquid used for washing in the present invention is an optimized washing liquid containing 0.05% Tween, which can reduce the interference background caused by the non-specific adsorption of the micropipe.

As a preferred technical solution, the detection method of the present invention comprises the following steps:

(1) Fabrication of the detection chip: the chip is bonded to the solid-phase reaction substrate to form a closed microfluidic channel, and the specific recombinant protein antigens of Mycoplasma pneumoniae and influenza virus are introduced into the microfluidic channel respectively. The recombinant protein antigen is coated on the substrate; after coating, the coating solution is drawn out, the chip is removed, and another chip is pasted, so that the pipes on the two chips are perpendicular to each other, and the crossing angle is 90°. After it is sealed with the substrate, the BSA sealing solution is passed into the microfluidic channel, and the sealing solution is drawn out after sealing;

(2) adding serum to the microfluidic pipeline sample hole in the detection chip described in step (1), incubating for 15 min, and washing with an optimized washing solution containing 0.05% Tween;

(3) adding the optimized HRP-labeled goat anti-human IgM antibody to the microfluidic pipeline described in step (2), washing with an optimized washing solution containing 0.05% Tween;

(4) Adding a luminol chemiluminescence substrate into the microfluidic pipeline described in step (3), and detecting it by a chemiluminescence detector.

Detection principle of the present invention is:

In the prepared detection chip, after the sample to be tested is added to the sample hole of the microchannel, the IgM antibody in the serum sample filled with the microchannel will bind to the antigen of the specific pathogen pre-coated on the substrate, and incubate for 15 minutes After washing, if the tested sample contains IgM specific to the specific antigen of Mycoplasma pneumoniae or influenza virus, then the specific IgM in the sample will bind to the antigen coated on the reaction area, and an immune reaction will occur to form the antigen-IgM Complex; then add enzyme-labeled detection antibody to all pipelines, if the detection sample contains specific IgM against Mycoplasma pneumoniae or influenza virus, then the enzyme-labeled detection antibody will be immunoimmunized with the specific IgM antibody bound to the reaction area react to form an antigen-IgM antibody-enzyme-labeled antibody conjugate; finally pass through the luminescent liquid to catalyze the reaction with the enzyme and generate a luminescent signal, which will be detected by the chemiluminescence analyzer and given a value. If there is no IgM specific for these two pathogens in the test sample, there will be no enzyme-labeled detection antibody bound to the antigen on the substrate, and finally no chemiluminescent signal will be detected.

In the present invention, in step (1), the specific recombinant protein antigen of Mycoplasma pneumoniae can be passed into the two columns of pipelines on the left side of the chip, and the specific recombinant protein of influenza virus can be passed into the two columns of pipelines on the right side of the chip. Antigen, which can realize the simultaneous detection of Mycoplasma pneumoniae and influenza virus.

If the injected sample produces luminescent signals in the two columns on the left side of the chip, and the value of the luminescent signal read by the chemiluminescence analyzer is greater than the cutoff value set according to the negative control, it indicates that the sample contains specific Mycoplasma pneumoniae IgM; The sample generates luminescent signals in the two columns on the right, and the reading value of the instrument is greater than the set cutoff value, indicating that the sample contains specific influenza virus IgM. If the reading value of the instrument is greater than the set cutoff value, it means that both Mycoplasma pneumoniae and influenza virus-specific IgM are contained; if the input sample does not produce luminescence signals at the corresponding positions on the left and right sides of the chip, it means that it does not contain Mycoplasma pneumoniae and influenza virus-specific IgM. Regardless of the condition of the negative part, as long as the positive quality control part in the first row above does not show a luminescent signal, the chip is judged to be invalid and needs to be tested again.

In the second aspect, the present invention also provides the application of the method described in the first aspect in the detection of pneumonia and influenza.

Compared with the prior art, the present invention has at least the following beneficial effects:

1. The present invention can jointly detect Mycoplasma pneumoniae and/or influenza virus-specific IgM in multiple samples by means of a microfluidic chip and select serum as a sample through a one-time operation. It has the advantages of small sample size, multiple samples, and multiple indicators. Detection, strong specificity, high sensitivity, low price, quantitative and intuitive reading results, etc.;

2. The present invention shortens the antigen fixation time and the antigen-antibody reaction time respectively. In some embodiments, the reaction time can be shortened to within 15 minutes; and the whole immune reaction, including antigen fixation, antibody reaction, and secondary antibody reaction, is shortened to Within 30 minutes, rapid detection is realized;

3. The present invention does not require complex instruments and equipment, nor does it require the experience and judgment of experimenters, and avoids the subjective factors of different operators. The overall coincidence rate of the test results is high, and it is suitable for on-site testing.

Description of drawings

Fig. 1 is a schematic diagram of detection chip production and detection process of the present invention;

Among them, steps ①-② are schematic diagrams for making the detection chip; steps ③-④ are schematic diagrams for the detection process;

Fig. 2 is the composite diagram of detection result judging principle of the present invention;

Among them, Figure 2a is a schematic diagram of specific pathogen antigen coating on the substrate when no sample is added; Figure 2b-e is a schematic diagram of the detection results after adding Mycoplasma pneumoniae and influenza virus samples at the same time, and Figure 2b is positive for Mycoplasma pneumoniae, influenza virus Virus negative, Figure 2c is negative for Mycoplasma pneumoniae, positive for influenza virus, Figure 2d is positive for Mycoplasma pneumoniae, positive for influenza virus, Figure 2e is negative for Mycoplasma pneumoniae, negative for influenza;

Fig. 3 is a schematic diagram of the results of simultaneous detection of Mycoplasma pneumoniae and influenza samples by the present invention;

Fig. 4 is the result figure of simultaneous detection of lung branch and influenza of the present invention;

Fig. 5 is the result figure that the pulmonary branch of the present invention detects alone;

Fig. 6 is the result figure that the influenza of the present invention detects alone;

Among them: 1-liquid inlet hole, 2-base, 3-pipe cavity, 4-liquid outlet hole.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

The reagent equipment source used in the embodiment:

(1) Mycoplasma pneumoniae recombinant protein: Xiamen Wankelong Biotechnology Co., Ltd.;

(2) Influenza virus recombinant protein: Yiqiao Sino Biotechnology Co., Ltd.;

(3) Mouse/goat anti-human IgM-HRP antibody, bovine serum albumin, mouse/goat anti-human IgG antibody: Beijing Suolaibao Biotechnology Co., Ltd.;

(4) PS substrate: corning;

(5) PDMS chip matrix, curing agent: Dow Corning;

(6) High temperature drying oven: Shanghai Yiheng Technology Co., Ltd.;

(7) PBS: Beijing Chemical Co., Ltd.;

(8) Chemiluminescent liquid: milipore;

(9) Chemiluminescence instrument: developed by National Center for Nanoscience and Technology

Simultaneous detection of embodiment 1 mycoplasma pneumoniae and influenza virus specific antibody IgM

(1) Preparation of detection chip

Take a multi-channel chip and seal it with the substrate, then take 1 μL of Mycoplasma pneumoniae recombinant protein stock solution and add it to PBS (PH7.2-7.4) buffer solution, dilute to the optimal coating concentration of 20 μg/mL, and mix well; the diluted protein The solution is passed into the two pipelines on the left; take 1 μL of influenza virus recombinant protein stock solution and add it to PBS (PH7.2-7.4) buffer solution, dilute to the optimal coating concentration of 15 μg/mL, and mix well; the diluted protein solution Pass into the two pipelines on the right; room temperature, coating for 30min.

Prepare 3% BSA blocking solution with PBS buffer. Remove the first chip, after air drying, paste the second chip, make the pipeline on the second chip perpendicular to the pipeline placement direction of the first chip, and seal; then use a pipette to inject 20 μL BSA into the pipeline Blocking solution at room temperature for 30 minutes; take out the blocking solution, dry it, put it in a sealed bag, and store it at 4°C to prepare the detection chip, as shown in steps ①-② in Figure 1.

(2) Use the above-mentioned detection chip to simultaneously detect the serum samples of Mycoplasma pneumoniae and influenza virus

① Collection of actual serum samples (note: the serum needs to be clarified, if it is turbid, high-speed centrifugation is required to remove turbid impurities);

② Treatment of serum samples: Take 1 μL of serum, use the purchased IgG adsorbent sample diluent to dilute the serum at a dilution concentration of 1:10, mix well, and wait for 10 minutes at room temperature before use;

③Start the test, open the sealed bag, take out the prepared chip, place the chip tube in parallel on the detection table, then use a pipette to take 20 μL of the sample to be tested, align it with the sample hole, pass it into the tube, incubate at room temperature for 15-20 minutes, and then take it out Test samples, wash with washing solution 4-5 times;

④Introduce 20 μL of detection antibody into each tube, incubate at room temperature for 15-20 minutes, take out the detection sample, and wash with washing solution 4-5 times;

⑤ Pass commercialized chemiluminescent liquid into the chip, put it into the chemiluminescent instrument, and use analysis software for data analysis;

⑥Result judgment: The detection principle is shown in Figure 2a-e, and the specific detection result judgment is shown in Figure 3 and Figure 4. After the sample is passed through, luminous signals are generated at the corresponding positions of the four columns on the left and right of the chip, and the instrument reads A value greater than the set cutoff value indicates that the sample contains both Mycoplasma pneumoniae and influenza virus-specific IgM.

The clinically confirmed positive serum and negative serum were detected by using the simultaneous detection chip of Mycoplasma pneumoniae and influenza virus according to the present invention, and the results are shown in Table 1.

Table 1

As can be seen from Table 1, after testing, the coincidence rate of the detection results for the positive samples of Mycoplasma pneumoniae can reach 96%, and the coincidence rate of the detection results for the positive samples of influenza virus can reach 100%, and there is no cross-reaction between the two during the detection process. , indicating that the overall coincidence rate of the test results is high, and the simultaneous detection of Mycoplasma pneumoniae and influenza virus-specific antibody IgM has been realized.

The detection of embodiment 2 Mycoplasma pneumoniae specific antibody IgM

(1) Preparation of detection chip

Take 1 μL of Mycoplasma pneumoniae recombinant protein and add it to PBS (pH7.2-7.4) buffer solution, dilute to the optimal coating concentration of 20 μg/mL, and mix well; take a multi-channel chip and seal it with the substrate; dilute the protein solution by The sample hole was connected to two pipelines, and was coated for 30 minutes at room temperature.

Prepare 3% BSA blocking solution with PBS buffer. Remove the first chip, after air drying, paste the second chip, make the pipeline on the second chip perpendicular to the pipeline placement direction of the first chip, and seal; then use a pipette to inject 20 μL BSA into the pipeline The blocking solution was kept at room temperature for 30 minutes; the blocking solution was taken out, dried, put into a sealed bag, and stored at 4°C to prepare a detection chip for Mycoplasma pneumoniae.

(2) Use the above-mentioned detection chip to detect the actual serum sample of Mycoplasma pneumoniae

①Collect the actual serum sample (Note: the serum needs to be clarified, if it is turbid, it needs to be centrifuged at a high speed to remove the turbid impurities);

② Treatment of serum samples: Take 1 μL of serum, use the purchased IgG adsorbent sample diluent to dilute the serum at a dilution concentration of 1:10, mix well, and wait for 10 minutes at room temperature before use;

③ Start the test, open the sealed bag, take out the prepared detection chip, place the chip tube in parallel on the detection table, then use a pipette to take 20 μL of the prepared sample to be tested and align it with the sample hole, pass it into the tube, and incubate at room temperature for 15 -Take out the test sample after 20 minutes, and wash with washing solution 4-5 times;

④Inject 20 μL of enzyme-labeled detection antibody into each tube, incubate at room temperature for 15-20 minutes, take out the detection sample, and wash with washing solution 4-5 times;

⑤ Pass commercialized chemiluminescent liquid into the chip, put it into the chemiluminescent instrument, and use analysis software for data analysis;

⑥ Judgment of results: The detection results are shown in Figure 5. After the sample is passed through, a luminescence signal is generated on the chip. The value of the luminescence signal read by the chemiluminescence analyzer is greater than the cutoff value set according to the negative control, indicating that the sample contains specific Mycoplasma pneumoniae IgM.

The clinically confirmed positive serum and negative serum were detected by using the Mycoplasma pneumoniae detection chip of the present invention, and the results are shown in Table 2.

Table 2

It can be seen from Table 2 that, after testing, the coincidence rate of the test results for positive samples can reach 97%, and the coincidence rate of test results for negative samples is 100%, indicating that the overall coincidence rate of test results is relatively high.

The detection of embodiment 3 influenza virus specific antibody IgM

(1) Preparation of detection chip

Take 1 μL of influenza virus recombinant protein and add it to PBS (PH7.2-7.4) buffer solution, dilute to the optimal coating concentration of 15 μg/mL, mix well; take a multi-channel chip and seal it with the substrate; dilute the protein solution by The sample hole was connected to two pipelines, and was coated for 30 minutes at room temperature.

Prepare 3% BSA blocking solution with PBS buffer. Remove the first chip, after air drying, paste the second chip, make the pipeline on the second chip perpendicular to the pipeline placement direction of the first chip, and seal; then use a pipette to inject 20 μL BSA into the pipeline Blocking solution at room temperature for 30 minutes; take out the blocking solution, dry it, put it into a sealed bag, and store it at 4°C to prepare the detection chip.

(2) Use the above-mentioned detection chip to detect the actual serum samples of influenza

① Collection of actual serum samples (note: the serum needs to be clarified, if it is turbid, high-speed centrifugation is required to remove turbid impurities);

② Treatment of serum samples: Take 1 μL of serum, use the purchased IgG adsorbent sample diluent to dilute the serum at a dilution concentration of 1:10, mix well, and wait for 10 minutes at room temperature before use;

③Start the test, open the sealed bag, take out the prepared chip, place the chip tube in parallel on the detection table, then use a pipette to take 20 μL of the sample to be tested, align it with the sample hole, pass it into the tube, incubate at room temperature for 15-20 minutes, and then take it out Test samples, wash with washing solution 4-5 times;

④Introduce 20 μL of detection antibody into each tube, incubate at room temperature for 15-20 minutes, take out the detection sample, and wash with washing solution 4-5 times;

⑤ Pass commercialized chemiluminescent liquid into the chip, put it into the chemiluminescent instrument, and use analysis software for data analysis;

⑥ Judgment of results: The test results are shown in Figure 6. After the sample is passed through, a luminescence signal is generated on the chip. The value of the luminescence signal read by the chemiluminescence analyzer is greater than the cutoff value set according to the negative control, indicating that the sample contains influenza. Virus IgM.

The clinically confirmed positive serum and negative serum were detected by using the influenza virus detection chip of the present invention, and the results are shown in Table 3.

table 3

It can be seen from Table 3 that after testing, the coincidence rate of the test results for positive samples can reach 98%, and the coincidence rate of test results for negative samples is 100%, indicating that the overall coincidence rate of test results is relatively high.

As can be seen from Examples 1-3, the detection method of the present invention can be used to detect the specific antibody IgM of Mycoplasma pneumoniae and influenza virus alone or simultaneously, and the detection result can be obtained through a one-time operation, which has the advantages of small sample size, Multiple indicators can be detected at the same time, strong specificity, high sensitivity, quantitative reading results, intuitive, fast, etc., and the overall coincidence rate of the test results is high, which has important clinical significance.

The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. a method for detecting the specific antibody IgM of Mycoplasma pneumoniae and influenza virus based on microfluidic chip, it is characterized in that, may further comprise the steps: (1) Fabrication of the detection chip: the chip is attached to the solid-phase reaction substrate to form a closed microfluidic channel, and the specific recombinant protein antigen of Mycoplasma pneumoniae and/or influenza virus is introduced into the microfluidic channel, and the The specific recombinant protein antigen is coated on the substrate; after coating, the coating solution is drawn out, the chip is peeled off, and another chip is pasted, so that the channels on the two chips cross each other, and after sealing it with the substrate , pass the blocking liquid into the micro-flow channel, and draw out the blocking liquid after sealing; (2) adding the sample to be tested to the microfluidic pipeline sample hole in the detection chip described in step (1), incubating and washing; (3) adding an enzyme-labeled detection antibody to the microfluidic pipeline described in step (2), and washing; (4) Adding chemiluminescence liquid into the microfluidic pipeline described in step (3), and detecting by a chemiluminescence detector. 2. The method according to claim 1, wherein the pipelines on the two chips described in step (1) are perpendicular to each other, and the angle of intersection is 90°; Preferably, the chip is a chip with parallel microfluidic channels through manual overmolding or injection molding; Preferably, the chip has 7 parallel microfluidic channels; preferably, the length of the microfluidic channels is 3-5cm, the diameter of the microfluidic channels is 500-600 μm, and the height is 500-600 μm. The spacing is 1-3mm. 3. The method according to claim 1 or 2, characterized in that, the solid-phase reaction substrate described in step (1) is polystyrene, polydimethylsiloxane or polymethyl methacrylate, preferably poly styrene. 4. The method according to any one of claims 1-3, characterized in that the tested sample in step (2) is serum. 5. The method according to any one of claims 1-4, characterized in that the incubation time in step (2) is 10-20 min, preferably 15 min. 6. The method according to any one of claims 1-5, characterized in that the enzyme-labeled detection antibody in step (3) is an optimized HRP-labeled goat anti-human IgM antibody. 7. The method according to any one of claims 1-6, characterized in that the chemiluminescent liquid in step (4) is a luminol chemiluminescent substrate. 8. The method according to any one of claims 1-7, characterized in that the washing liquid used for washing is an optimized washing liquid containing 0.05% Tween. 9. The method according to any one of claims 1-8, comprising the steps of: (1) Fabrication of the detection chip: the chip is bonded to the solid-phase reaction substrate to form a closed microfluidic channel, and the specific recombinant protein antigens of Mycoplasma pneumoniae and influenza virus are introduced into the microfluidic channel respectively. The recombinant protein antigen is coated on the substrate; after the coating is completed, the coating solution is drawn out, the chip is removed, and another chip is pasted, so that the pipes on the two chips are perpendicular to each other, and the crossing angle is 90°. After it is sealed with the substrate, the BSA sealing solution is passed into the microfluidic channel, and the sealing solution is drawn out after sealing; (2) adding serum to the microfluidic pipeline sample hole in the detection chip described in step (1), incubating for 15 min, and washing with an optimized washing solution containing 0.05% Tween; (3) adding the optimized HRP-labeled goat anti-human IgM antibody to the microfluidic pipeline described in step (2), washing with an optimized washing solution containing 0.05% Tween; (4) Adding a luminol chemiluminescence substrate into the microfluidic pipeline described in step (3), and detecting it by a chemiluminescence detector. 10. Application of the method according to any one of claims 1-9 in the detection of pneumonia and influenza.