Mycoplasma pneumoniae detection test strip based on high-concentration colloidal gold and preparation method thereof

Abstract

The preparation method of the mycoplasma pneumoniae detection test strip based on the high-concentration colloidal gold comprises the following steps: preparing a labeled reconstitution fluid, respectively reacting a mycoplasma pneumoniae detection antibody, a chicken IgY antibody and a colloidal gold solution with the concentration of OD100 to prepare a reconstitution fluid, mixing and spraying the reconstitution fluid on a conjugate pad; a detection line and a quality control line are arranged on the coating film, a corresponding antibody solution is sprayed, finally, the sample pad, the combination pad, the coating film and the water absorption pad are assembled to obtain the test strip, in addition, the invention further relates to the steps of pretreatment of the sample pad and the combination pad, addition of a buffer solution in a colloidal gold solution and the like. The invention also provides a test strip prepared by the method and a lung cancer auxiliary diagnosis product containing the test strip, and the test strip and the lung cancer auxiliary diagnosis product have the advantages of simplicity and convenience in operation, rapidness, sensitivity and the like, can be effectively used for detecting mycoplasma pneumoniae, and provide powerful support for clinical diagnosis.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, in particular to a Mycoplasma pneumoniae detection test strip based on high-concentration colloidal gold and a preparation method thereof. Background Art

[0002] In the field of respiratory diseases, Mycoplasma pneumoniae is an extremely common pathogen, especially in children and adolescents, where the infection rate is quite high, and has become one of the key pathogenic factors of respiratory infections in this age group. This microorganism has a unique cell structure, and its most prominent feature is the lack of a cell wall, which makes Mycoplasma pneumoniae naturally resistant to many conventional antibiotics. Therefore, rapid and accurate diagnosis is particularly critical when dealing with Mycoplasma pneumoniae infection. Only by timely and accurate determination of the infection status can we lay a solid foundation for subsequent effective treatment and epidemic prevention and control, and prevent the disease from worsening or spreading further due to delayed diagnosis.

[0003] Traditional methods of detecting Mycoplasma pneumoniae include serological tests, PCR technology, and culture methods. Serological tests are mainly based on detecting specific antibodies produced in the patient's body due to Mycoplasma pneumoniae infection, and inferring whether the infection is present by the presence of antibodies in blood samples and changes in titers. This detection method is extremely complicated to operate, requiring the collection of patient blood, followed by a series of complex laboratory operation procedures. The entire process is time-consuming, and it usually takes hours or even days to obtain preliminary results. In addition, the accuracy of serological tests is easily affected by a variety of factors, such as the time stage of the patient's infection, differences in individual immune responses, etc. These factors may lead to false positive or false negative test results.

[0004] Polymerase chain reaction technology (PCR) has extremely high sensitivity and specificity, and can detect extremely small amounts of pathogen nucleic acid. However, PCR technology has extremely high requirements for laboratory equipment and the professional qualities of technicians. It requires rigorous professional training and proficiency in various complex technical links. At the same time, the PCR detection process involves many steps, including sample collection, transportation, storage, nucleic acid extraction, amplification reaction, and result determination. Each link requires strict quality control, which can easily lead to inaccurate results. Coupled with the high cost of PCR testing, factors such as reagent costs, equipment maintenance costs, and labor costs make it difficult for this detection method to be widely promoted and used in primary medical institutions, thereby limiting its popularity in clinical diagnosis.

[0005] The culture method is a traditional and classic way to detect Mycoplasma pneumoniae. It is to inoculate the patient sample into a specific culture medium, cultivate it under a suitable culture environment and conditions for a period of time, and then check whether Mycoplasma pneumoniae grows. However, the disadvantage of this method is that the culture method is time-consuming, and it usually takes several days to weeks to get accurate culture results, which is unacceptable for patients who are in urgent need of timely diagnosis and treatment. In addition, the culture method has extremely strict requirements on the culture environment and conditions. It requires precise control of many factors such as the composition, pH value, temperature, humidity, etc. of the culture medium, and it is also necessary to prevent contamination by other bacteria, otherwise it is very easy to cause culture failure.

[0006] In recent years, immunochromatography has been widely used in on-site screening of various infectious diseases due to its simplicity and speed. However, existing Mycoplasma pneumoniae immunochromatography test strips still have some shortcomings in terms of sensitivity, specificity and stability. In addition, conventionally used markers such as ordinary colloidal gold particles may not provide sufficient signal intensity in some cases, resulting in unstable or misjudgment of test results. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a Mycoplasma pneumoniae detection test strip based on high-concentration colloidal gold and a preparation method thereof. The Mycoplasma pneumoniae detection test strip consists of a base plate, a sample pad, a conjugation pad, a coating film and a water absorbent pad. The coating film is provided with a Mycoplasma pneumoniae detection line and a quality control line. High-concentration colloidal gold is used to label Mycoplasma pneumoniae detection antibodies. By optimizing the antibody loading and other steps in the preparation process, a colloidal gold detection test strip with higher sensitivity, higher specificity and stronger colloidal gold particle signal intensity is prepared.

[0008] The first object of the present invention is to provide a method for preparing a Mycoplasma pneumoniae test strip based on high-concentration colloidal gold, which specifically comprises the following steps:

[0009] (1) preparing a colloidal gold labeling complex solution;

[0010] (2) adding the Mycoplasma pneumoniae detection antibody to a high concentration colloidal gold solution to make the final concentration of the detection antibody 50-80 μg / mL, adding 0.6-1 wt% bovine serum albumin, centrifuging to obtain a precipitate, adding a colloidal gold labeled reconstitution solution to the precipitate, and obtaining a colloidal gold labeled Mycoplasma pneumoniae detection antibody reconstitution solution, wherein the concentration of the high concentration colloidal gold is OD100;

[0011] (3) adding chicken IgY antibody to the colloidal gold solution to make the final concentration of the chicken IgY antibody be 50-80 μg / mL, adding 0.6-1 wt % bovine serum albumin, centrifuging to obtain a precipitate, adding colloidal gold labeled reconstitution solution to the precipitate, and obtaining a colloidal gold labeled chicken IgY antibody reconstitution solution;

[0012] (4) mixing the reconstituted solutions obtained in step (2) and step (3) to obtain a mixed reconstituted solution, and spraying the mixed reconstituted solution on a conjugate pad;

[0013] (5) Setting a test line and a quality control line on the coating membrane, spraying a solution containing Mycoplasma pneumoniae capture antibody and a solution containing goat anti-chicken IgY antibody respectively, wherein the test line and the quality control line are arranged perpendicular to the direction of the test strip, the final concentration of the Mycoplasma pneumoniae capture antibody is 0.6-1 mg / mL, and the solution containing the Mycoplasma pneumoniae capture antibody is sprayed on the test line at 1-2 μL / cm;

[0014] (6) Assembling a sample pad, a conjugate pad, a coating membrane and a water-absorbing pad to obtain the Mycoplasma pneumoniae test strip.

[0015] Furthermore, the method further comprises the step of pretreating the sample pad and the conjugate pad, wherein the pretreating step is to soak the sample pad and the conjugate pad in a sample pad pretreating solution and a conjugate pad pretreating solution respectively;

[0016] The components of the sample pad pretreatment solution include buffer, bovine serum albumin, polyvinyl pyrrolidone, surfactant, preservative and blocking agent;

[0017] The components of the binding pad pretreatment solution include buffer, bovine serum albumin, polyvinyl pyrrolidone, surfactant and preservative.

[0018] Further, the sample pad pretreatment solution includes 40-80mM Tris-HCl, 0.4-0.8wt% casein or bovine serum albumin, 0.8-1.5wt% polyvinylpyrrolidone K30, 0.8w-1.5wt% surfactant S9, 0.04-0.1wt% Tween 20, 0.02-0.05wt% sodium azide or ProClin300 and 0.002-0.006wt% heterophilic antibody blocking agent HBR.

[0019] Furthermore, the pH value of the sample pad pretreatment solution is 7-8.

[0020] Furthermore, the conjugate pad pretreatment solution includes 40-80 mM Tris-HCl, 0.4-0.8 wt % casein or BSA, 0.8-1.5 wt % polyvinyl pyrrolidone K30, 0.04-0.08 wt % Tween 20, and 0.02-0.06 wt % sodium azide or ProClin300.

[0021] Furthermore, the pH value of the conjugate pad pretreatment solution is 8-8.5.

[0022] Furthermore, a labeling buffer is added to the colloidal gold solution in step (1), wherein the labeling buffer is a boric acid-borax buffer and the pH value of the labeling buffer is 7.3-7.6.

[0023] Furthermore, the concentration of the boric acid-borax buffer is 0.01M.

[0024] Furthermore, in step (2), the amount of the colloidal gold labeling solution added is 70-80 μL.

[0025] Furthermore, in step (3), the amount of the colloidal gold labeling solution added is 90-100 μL.

[0026] Furthermore, in step (4), 70-80 μL of colloidal gold-labeled Mycoplasma pneumoniae detection antibody reconstitution solution and 20-25 μL of colloidal gold-labeled chicken IgY antibody reconstitution solution are mixed.

[0027] Furthermore, the spraying amount of the mixed solution is 2-4 μL / cm.

[0028] Furthermore, the final concentration of the goat anti-chicken IgY antibody is 0.8-1.2 mg / mL, and the solution containing the goat anti-chicken IgY antibody is sprayed on the quality control line at 1-2 μL / cm.

[0029] The second object of the present invention is to provide a Mycoplasma pneumoniae test strip prepared by the above method.

[0030] Furthermore, the Mycoplasma pneumoniae test strip also includes a sample diluent.

[0031] Furthermore, the sample diluent includes 1-1.5wt% Tris, 0.5-1wt% bovine serum albumin or casein, 0.5-1wt% surfactant S22, and 0.03-0.06wt% sodium azide or ProClin300.

[0032] Furthermore, the pH value of the sample diluent is 7-7.5.

[0033] The third object of the present invention is to provide a lung cancer auxiliary diagnosis product comprising the above-mentioned Mycoplasma pneumoniae test strip.

[0034] Beneficial effects of the present invention:

[0035] The present invention prepares the Mycoplasma pneumoniae test strip by adopting high concentration colloidal gold (OD100), which significantly improves the detection performance. High concentration colloidal gold makes the detection signal stronger, greatly enhances the sensitivity of the test strip, can more accurately capture the trace of Mycoplasma pneumoniae, and effectively reduces the risk of missed detection. Under the synergistic effect of the optimized antibody concentration and other components, not only the high specificity of the detection is guaranteed, false positive and false negative results are avoided, but also the stability and repeatability of the test strip are improved. The simplicity of its operation is not limited by the environment and the professional degree of the personnel, and it has obvious advantages in primary medical units and on-site detection, and can quickly provide reliable test results, which plays a key role in the early diagnosis and prevention and control of Mycoplasma pneumoniae infection, and effectively promotes the progress of pneumonia disease diagnosis technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0037] Figure 1 Schematic diagram of the structure of the Mycoplasma pneumoniae test strip based on high-concentration colloidal gold in Example 1 of the present invention, wherein 1 is a sample pad, 2 is a binding pad, 3 is a nitrocellulose membrane, 31 is a detection line, 32 is a quality control line, 4 is a water-absorbing pad, and 5 is a bottom plate;

[0038] Figure 2 The results of the test strips in Example 2 of the present invention are compared, wherein 1 is the concentration of Mycoplasma pneumoniae antigen and 10 4 CFU / mL test result, color development result is positive; 2 is the concentration of Mycoplasma pneumoniae antigen 10 2 The CFU / mL test result showed that the color development result was positive; 3 and 4 were the test results of Mycoplasma pneumoniae antigen concentrations of 10 CFU / mL and 0 CFU / mL, respectively, and both showed negative results. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Example 1: Preparation method of test strips

[0041] 1. Pre-treat the sample pad

[0042] Sample pad treatment solution: 50 mM Tris-HCl, 0.6 wt % casein, 0.8 wt % polyvinylpyrrolidone K30, 1 wt % surfactant S9, 0.05 wt % Tween 20, 0.05 wt % ProClin 300 and 0.004 wt % heterophilic antibody blocker HBR, solution pH = 7.5.

[0043] The sample pad treatment solution was added at 0.08 mL / cm 2 Add the amount of into the soaking container, soak the sample pad for 5 minutes, dehydrate it and place it in a 45℃ oven to dry for 7 hours, and store it in a sealed state under a humidity ≤30%.

[0044] 2. Pre-treat the conjugate pad

[0045] Conjugate pad treatment solution: 50 mM Tris-HCl, 0.5 wt % BSA, 1 wt % polyvinylpyrrolidone K30, 0.05 wt % Tween 20, 0.05 wt % ProClin 300, solution pH = 8.5.

[0046] The conjugate pad treatment solution was added at 0.06 mL / cm 2 Add the amount of into the soaking container, soak the conjugate pad for 5 minutes, place the dehydrated conjugate pad in a 45°C oven for 7 hours, and then place the dried conjugate pad in an environment with a humidity of ≤30% and seal it for storage.

[0047] 3. Labeling and reconstitution of mouse anti-human Mycoplasma pneumoniae detection antibodies

[0048] High concentration colloidal gold: particle size 60nm, OD100.

[0049] Labeling buffer: 0.01 M boric acid-borax buffer, pH = 7.4.

[0050] Colloidal gold labeling solution: 50 mM Tris-HCl, 0.6 wt % BSA, 15 wt % sucrose, 5 wt % trehalose, 0.05 wt % ProClin 300, solution pH = 8.

[0051] 40 μL of high-concentration colloidal gold was added to 60 μL of labeling buffer, and then mouse anti-human pneumonia Mycoplasma detection antibody was added to make its concentration 60 μg / mL, followed by mixing and reacting for 1.5 hours; bovine serum albumin BSA was added to make its final concentration 1wt%, mixed and reacted for 20 minutes; the solution was subjected to low-temperature high-speed centrifugation, the centrifugation parameters were temperature 4°C, rotation speed 8000 rpm, centrifugation for 5 minutes, and the supernatant was removed; 80 μL of colloidal gold labeling reconstitution solution was added to the precipitate, and then the precipitate was broken up and resuspended to obtain a colloidal gold-labeled mouse anti-human pneumonia Mycoplasma detection antibody reconstitution solution.

[0052] 4. Labeling and reconstitution of chicken IgY antibody

[0053] Take 40 μL of high-concentration colloidal gold, add 60 μL of 0.01M boric acid-borax buffer, add chicken IgY antibody to make its concentration 60 μg / mL, then mix and react for 1.5 hours; add BSA to make its concentration 0.6-1wt%, then mix and react for 20 minutes; low-temperature high-speed centrifugation, the centrifugation parameters are temperature 4°C, rotation speed 8000rpm, centrifugation for 5 minutes, and remove the supernatant; add 100 μL of gold-labeled rehydration solution to the precipitate, then break up and resuspend the precipitate to obtain a colloidal gold-labeled chicken IgY antibody rehydration solution.

[0054] 5. Capture Antibody Coating

[0055] Coating diluent: 10 mM PBS, 1.5 wt% sucrose, solution pH = 7-7.5.

[0056] The mouse anti-human pneumonia Mycoplasma capture antibody was diluted to 0.8 mg / mL with a coating diluent to obtain a mouse anti-human pneumonia Mycoplasma capture antibody coating solution; the goat anti-chicken IgY antibody was diluted to 1 mg / mL to obtain a goat anti-chicken IgY antibody coating solution.

[0057] 6. Assembly

[0058] The gold-labeled complex solution is sprayed on the conjugate pad by mixing 80 μL of colloidal gold-labeled mouse anti-human mycoplasma pneumoniae detection antibody complex solution and 20 μL of colloidal gold-labeled chicken IgY antibody complex solution evenly, and spraying it on the treated conjugate pad at a spray volume of 3 μL / cm, then baking the sprayed conjugate pad at 45° C. for 6 hours, and then placing the dried conjugate pad in an environment with a humidity of ≤30% and sealing it for storage;

[0059] The coating solution is sprayed on the nitrocellulose membrane, and the mouse anti-human pneumonia Mycoplasma capture antibody coating solution and the goat anti-chicken IgY antibody coating solution are sprayed on the nitrocellulose membrane at a spray volume of 1 μL / cm to form a detection line and a quality control line, and then the treated nitrocellulose membrane is placed at 45° C. and baked for 6 hours, and then the dried nitrocellulose membrane is placed in an environment with a humidity of ≤30% and sealed for storage;

[0060] The positional relationship between the sample pad, the conjugate pad, the nitrocellulose membrane, the water-absorbing pad and the bottom plate is as follows: the sample pad, the conjugate pad, the water-absorbing pad and the nitrocellulose membrane are all bonded to the bottom plate, wherein the nitrocellulose membrane is located near the middle of the bottom plate, one end of the nitrocellulose membrane is bonded to the water-absorbing pad with a 2 mm overlap, the other end of the nitrocellulose membrane is bonded to the conjugate pad with a 2 mm overlap, the other end of the conjugate pad is bonded to the sample pad with a 2 mm overlap, and the bottom plate is located at the bottom to support the entire test strip.

[0061] The test strip also includes a sample diluent, which consists of 1 wt% Tris, 0.8 wt% BSA or casein, 0.5 wt% surfactant S22, and 0.05 wt% ProClin300, with a solution pH of 7.2.

[0062] Embodiment 2: Test strip detection method

[0063] Use the sample diluent of Example 1 to dilute the concentration of 10 7 CFU / mL of Mycoplasma pneumoniae antigen standard was diluted to the following concentrations: 10 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, take 80 μL of the above five concentrations of antigen standard solution and sample diluent (Mycoplasma pneumoniae antigen concentration 0 CFU / mL), respectively, and add them to the test strip sample pad prepared in Example 1. Due to capillary action, the sample will move along the test strip to the conjugate pad and nitrocellulose membrane, and the result will be observed for 10 minutes (Note: color development is invalid after 15 minutes); take 4 representative concentrations of Mycoplasma pneumoniae antigen 0 CFU / mL, 10 CFU / mL, 10 2 CFU / mL and 10 4 The CFU / mL test results were compared. Figure 2 The Mycoplasma pneumoniae antigen concentration was greater than or equal to 10 2 The CFU / mL test result is positive, and the concentration is less than 10 2 The CFU / mL test result was negative.

[0064] Comparative Example 1: Adjustment of pH value of buffer solution

[0065] The test strips were prepared according to the method described in Example 1, except that: the pH value of the labeling buffer boric acid-borax buffer was adjusted to 8.4-9;

[0066] The Mycoplasma pneumoniae antigen standard solution was tested according to the method described in Example 2. The results showed that the concentration of Mycoplasma pneumoniae antigen was greater than or equal to 10 3 The CFU / mL test result is positive, and the concentration is less than 10 3 The CFU / mL test result was negative;

[0067] The test strips were prepared according to the method described in Example 1, except that: the pH of the labeling buffer boric acid-borax buffer was adjusted to 7.0-7.2;

[0068] The Mycoplasma pneumoniae antigen standard solution was tested according to the method described in Example 2. The results showed that the concentration of Mycoplasma pneumoniae antigen was greater than or equal to 10 3 The CFU / mL test result is positive, and the concentration is less than 10 3 The CFU / mL test result was negative;

[0069] It can be seen from the above results that the pH value of the labeled buffer will affect the sensitivity of the test strip. When the pH of the labeled buffer is too high or too low, the sensitivity of the test strip decreases.

[0070] Comparative Example 2: Adjustment of Antibody Loading

[0071] (1) A test strip was prepared according to the method described in Example 1, except that 160 μL of colloidal gold labeling reconstitution solution was used to reconstitute the colloidal gold-labeled mouse anti-human Mycoplasma pneumoniae detection antibody;

[0072] The Mycoplasma pneumoniae antigen standard solution was tested according to the method described in Example 2. The results showed that the concentration of Mycoplasma pneumoniae antigen was greater than or equal to 10 3 The CFU / mL test result is positive, and the concentration is less than 10 3 The CFU / mL test result was negative;

[0073] (2) A test strip was prepared according to the method described in Example 1, except that: an antibody coating solution was prepared and the mouse anti-human Mycoplasma pneumoniae capture antibody was diluted to 0.5 mg / mL;

[0074] The Mycoplasma pneumoniae antigen standard solution was tested according to the method described in Example 2. The results showed that the concentration of Mycoplasma pneumoniae antigen was greater than or equal to 10 3 The CFU / mL test result is positive, and the concentration is less than 10 3 The CFU / mL test result was negative;

[0075] From the above results, it can be seen that reducing the amount of detection antibody on the conjugate pad or the amount of capture antibody on the nitrocellulose membrane will reduce the detection performance of the test strip.

[0076] Comparative Example 3: Comparison of detection effect with commercially available test strips

[0077] The test strip prepared in Example 1 was compared with the commercially available test strip. The Mycoplasma pneumoniae antigen standard solution was tested on the test strip prepared in Example 1 and the commercially available test strip, and the results were observed after 10 minutes. The results are shown in Table 1.

[0078] Table 1 Comparison of different concentrations of Mycoplasma pneumoniae antigen standards

[0079]

[0080] From the above results, it can be seen that the Mycoplasma pneumoniae test strip provided by the present invention has a low Mycoplasma pneumoniae antigen concentration of 10 2 CFU / mL, it can still detect positive results, and the detection sensitivity is significantly improved compared with commercially available test strips.

[0081] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a Mycoplasma pneumoniae test strip based on high concentration colloidal gold, characterized in that: The specific steps include: (1) preparing a colloidal gold labeling complex solution; (2) adding the Mycoplasma pneumoniae detection antibody to a high concentration colloidal gold solution to make the final concentration of the detection antibody 50-80 μg / mL, adding 0.6-1 wt% bovine serum albumin, centrifuging to obtain a precipitate, adding a colloidal gold labeled reconstitution solution to the precipitate, and obtaining a colloidal gold labeled Mycoplasma pneumoniae detection antibody reconstitution solution, wherein the concentration of the high concentration colloidal gold solution is OD100; (3) adding chicken IgY antibody to the colloidal gold solution to make the final concentration of the chicken IgY antibody be 50-80 μg / mL, adding 0.6-1 wt % bovine serum albumin, centrifuging to obtain a precipitate, adding colloidal gold labeled reconstitution solution to the precipitate, and obtaining a colloidal gold labeled chicken IgY antibody reconstitution solution; (4) mixing the reconstituted solutions obtained in step (2) and step (3) to obtain a mixed reconstituted solution, and spraying the mixed reconstituted solution on a conjugate pad; (5) Setting a test line and a quality control line on the coating membrane, spraying a solution containing Mycoplasma pneumoniae capture antibody and a solution containing goat anti-chicken IgY antibody respectively, wherein the test line and the quality control line are arranged perpendicular to the direction of the test strip, the final concentration of the Mycoplasma pneumoniae capture antibody is 0.6-1 mg / mL, and the solution containing the Mycoplasma pneumoniae capture antibody is sprayed on the test line at 1-2 μL / cm; (6) Assembling a sample pad, a conjugate pad, a coating membrane and a water-absorbing pad to obtain the Mycoplasma pneumoniae test strip.

2. The preparation method according to claim 1, characterized in that: The step of pretreating the sample pad and the conjugate pad is also included, wherein the pretreating comprises soaking the sample pad and the conjugate pad in a sample pad pretreating solution and a conjugate pad pretreating solution respectively; The components of the sample pad pretreatment solution include buffer, bovine serum albumin, polyvinyl pyrrolidone, surfactant, preservative and blocking agent; The components of the binding pad pretreatment solution include buffer, bovine serum albumin, polyvinyl pyrrolidone, surfactant and preservative.

3. The preparation method according to claim 1, characterized in that: In step (1), a labeling buffer is further added to the colloidal gold solution. The labeling buffer is a boric acid-borax buffer and the pH value of the labeling buffer is 7.3-7.

6.

4. The preparation method according to claim 1, characterized in that: In step (2), the amount of the colloidal gold labeling solution added is 70-80 μL.

5. The preparation method according to claim 1, characterized in that: In step (3), the amount of the colloidal gold labeling solution added is 90-100 μL.

6. The preparation method according to claim 1, characterized in that: In step (4), 70-80 μL of colloidal gold-labeled Mycoplasma pneumoniae detection antibody reconstitution solution and 20-25 μL of colloidal gold-labeled chicken IgY antibody reconstitution solution are mixed.

7. The preparation method according to claim 6, characterized in that: The spraying amount of the mixed solution is 2-4 μL / cm.

8. The preparation method according to claim 1, characterized in that: In step (5), the final concentration of the goat anti-chicken IgY antibody is 0.8-1.2 mg / mL, and the solution containing the goat anti-chicken IgY antibody is sprayed on the quality control line at 1-2 μL / cm.

9. A Mycoplasma pneumoniae test strip prepared by the method of any one of claims 1 to 7.

10. A lung cancer auxiliary diagnosis product comprising the Mycoplasma pneumoniae test strip according to claim 8.

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