Mycoplasma bovis rapid detection method based on nano antibody

Abstract

The invention relates to the technical field of nano biosensing, in particular to a mycoplasma bovis rapid detection method based on a nano antibody, which comprises the following steps: expressing a mycoplasma bovis specific nano antibody by gene-edited escherichia coli, and culturing in a culture medium containing a novel protein expression accelerant and a self-assembly inducer to obtain the mycoplasma bovis rapid detection method based on the nano antibody. Chemical marking is carried out through clicking by a special fluorescent marker; treating a sample with a surfactant and a nuclease inhibitor, centrifuging, taking supernatant, and reacting with the labeled nano antibody and the signal amplifying agent in the micro-fluidic chip; magnetic separation is combined with time-resolved fluorescence immunoassay, and a fluorescence signal ratio is detected through dual-wavelength excitation, so that whether the mycoplasma bovis exists in a sample is judged. The detection method is rapid in detection and can be completed within one hour; the accuracy is high, and misjudgment is avoided; the operation is simple and convenient; cost is low and popularization is easy; and high throughput and universality are achieved.

Description

Technical Field

[0001] The present invention relates to the field of nanobiosensing technology, and particularly to a rapid detection method for Mycoplasma bovis based on nanobody. Background Art

[0002] Mycoplasma bovis disease is a serious infectious disease caused by Mycoplasma bovis, which can lead to bovine respiratory disease syndrome, mastitis, arthritis and other diseases, seriously affecting the growth, milk production and reproductive performance of cattle, and bringing huge economic losses to the global cattle industry. Rapid and accurate detection of Mycoplasma bovis is crucial for early diagnosis, prevention and control of diseases and epidemic situation control.

[0003] The detection methods of Mycoplasma bovis mainly include traditional culture method, serological detection method and molecular biology detection method. As the "gold standard" for detecting Mycoplasma bovis, the traditional culture method requires inoculating samples on specific media and culturing for several days or even weeks under suitable conditions, and identifying Mycoplasma bovis by observing the colony morphology and characteristics. This method is time-consuming, cumbersome in operation, and has high requirements for laboratory conditions and operator skills, making it difficult to meet the needs of rapid diagnosis. At the same time, due to the slow growth of Mycoplasma bovis and the possible interference of other microorganisms in the samples with the culture process, the detection sensitivity is relatively low, and false negative results are likely to occur.

[0004] Serological detection methods, such as enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination assay, etc., are based on the principle of antigen-antibody reaction for detection. Although these methods are relatively simple in operation and low in detection cost, they have the problem of insufficient specificity. There are antigen cross-reactions between Mycoplasma bovis and other mycoplasmas, which are likely to lead to false positive results; moreover, serological detection can only detect Mycoplasma bovis antibodies and cannot directly detect pathogens, so it cannot be used for early diagnosis of diseases, because in the early stage of infection, the body has not produced enough antibodies.

[0005] Molecular biology detection methods, represented by polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR, can directly detect the nucleic acid of Mycoplasma bovis, and have the advantages of high sensitivity, strong specificity and fast detection speed. However, this method has extremely high requirements for instrument equipment and operator professional skills, the detection cost is expensive, and it needs to be carried out in a professional laboratory environment, making it difficult to be popularized and applied in grass-roots farms and on-site rapid detection. In addition, problems such as primer dimers and template contamination are likely to occur during the PCR detection process, affecting the accuracy of the detection results.

[0006] With the development of nanobiotechnology, nanobodies have shown great potential in the field of biological detection due to their small molecular weight, high stability, strong specificity, etc. However, the existing mycoplasma bovis detection methods based on nanobodies still have many deficiencies, such as low expression efficiency of nanobodies, poor detection sensitivity and stability of markers, complex detection process, and unsatisfactory signal amplification effect. Therefore, there is an urgent need to develop an innovative, fast and accurate, easy-to-operate, and low-cost nanobody-based rapid detection method for mycoplasma bovis to meet the actual needs of the prevention and control of mycoplasma bovis disease. Summary of the Invention

[0007] (I) Technical problems to be solved In view of the deficiencies of the prior art, the present invention provides a rapid detection method for mycoplasma bovis based on nanobodies.

[0008] (II) Technical solutions A rapid detection method for mycoplasma bovis based on nanobodies comprises the following steps: S1: Preparation and modification of nanobodies. A gene fragment encoding a mycoplasma bovis-specific nanobody is introduced into an Escherichia coli expression system modified by CRISPR-Cas9 gene editing, and this system has knocked out genes related to lipopolysaccharide synthesis and inserted an enhanced green fluorescent protein regulatory element; in a culture medium containing a novel protein expression promoter Y with a mass concentration of 0.05%-0.1% and a self-assembly inducer X with a mass concentration of 0.03%-0.07%, culture at 37°C and 220-240 r / min for 10-12 hours; extract and purify the nanobodies, and label them with a novel fluorescent marker Z. S2: Sample pretreatment Take 1-5 mL of the sample to be detected, including bovine respiratory secretions and milk, add a surfactant W with a mass concentration of 0.2%-0.5% and a nuclease inhibitor N with a mass concentration of 0.01%-0.03%, oscillate and incubate at 32-34°C for 12-14 minutes, then centrifuge at 5000-5500 r / min for 7-9 minutes, and take the supernatant for use. S3: Immune reaction Add the labeled nanobodies obtained in step S1 to the supernatant of step S2, so that the final concentration of the labeled nanobodies is 2-4 μg / mL, and at the same time add a signal amplifier K with a mass concentration of 0.1%-0.3%, incubate at 26-28°C for 16-18 minutes to form an immune complex. S4: Detection and result determination The reaction system is detected by combining time-resolved fluorescence immunoassay and magnetic separation technology; the immune complex is separated by applying an external magnetic field, and the fluorescence signals of upconversion nanoparticles and quantum dots are detected by dual-wavelength excitation. When the ratio of the intensities of the two fluorescence signals exceeds the threshold set according to the standard curve, it is determined that Mycoplasma bovis is present in the sample; if the ratio is lower than the threshold, it is determined that Mycoplasma bovis is not present in the sample.

[0009] Preferably, during the expression of S1 nanobody, the metabolite concentration of the fermentation broth is monitored in real time. When the acetic acid concentration exceeds 0.5 g / L, a glucose solution with a mass concentration of 5%-10% and a mixed solution of amino acids with a mass concentration of 0.1%-0.3% are automatically added for metabolic regulation.

[0010] Preferably, in S3 immune reaction, a microfluidic chip is used for the reaction. The surface of the chip channel is modified with a capture antibody against the surface antigen of Mycoplasma bovis, and the flow rate of the reaction system in the chip is controlled at 5-10 μL / min.

[0011] Preferably, in the novel protein expression promoter Y, the molecular weight of the polyethylene glycol-polyethyleneimine-cyclodextrin terpolymer is 8000-12000 Da, the molar ratio of manganese ions to porphyrin in the manganese-porphyrin complex is 1:1, and its complexation degree is determined by ultraviolet-visible spectroscopy to be not less than 90%.

[0012] Preferably, the dendrimer generation of the self-assembly inducer X is 4-6 generations, and the degree of terminal thiolation is 70%-85%, and its structure is characterized by nuclear magnetic resonance hydrogen spectrum.

[0013] Preferably, in the signal amplifier K, the particle size of the magnetic nanoparticles in the streptavidin-alkaline phosphatase-magnetic nanoparticle conjugate is 20-30 nm, which is determined by transmission electron microscopy, and the binding ratio of the biotinylated auxiliary antibody to streptavidin is 4:1.

[0014] Preferably, during the time-resolved fluorescence immunoassay detection, the detection delay time of the fluorescence signal of the upconversion nanoparticles is 50-100 μs, and the detection window time of the fluorescence signal of the quantum dots is 10-20 ms.

[0015] Preferably, the kit for the rapid detection method of Mycoplasma bovis based on nanobody according to any one of the above includes reagent bottles containing freeze-dried labeled nanobody, surfactant W, nuclease inhibitor N, and signal amplifier K, as well as a standard Mycoplasma bovis antigen solution for calibration, and also includes a microfluidic chip and an operation guide for a portable fluorescence detector.

[0016] Preferably, the concentration of the lyophilized labeled nanoantibody after reconstitution is 10-30 μg / mL, the lyophilization protectant is composed of trehalose and mannitol in a mass ratio of 1:1, the pre-freezing temperature of the lyophilization process is -40°C, the vacuum degree is 15-20Pa, and the lyophilization time is 24-36 hours.

[0017] Preferably, the portable fluorescence detector has a built-in artificial intelligence algorithm, which can automatically analyze the fluorescence signal ratio and generate a test report, and the compliance rate of the test results with the laboratory standard method is not less than 95%.

[0018] 3. Beneficial technical effects Compared with the prior art, the present invention has the following beneficial effects: 1. By optimizing the reaction conditions of each step and adopting advanced technology, the entire detection process can be completed within 1 hour, which greatly shortens the detection time compared with the traditional culture method, and can gain precious time for the early diagnosis and timely prevention and control of bovine mycoplasma disease.

[0019] 2. The gene-edited Escherichia coli expression system and new protein expression promoter were used to improve the expression efficiency and specificity of nanoantibodies. The new fluorescent markers were combined with click chemistry reactions to enhance labeling stability. The signal amplifier and dual-wavelength excitation detection technology greatly improved the sensitivity and accuracy of detection, effectively avoiding false positive and false negative results. The compliance rate of the test results with the laboratory standard methods was no less than 95%.

[0020] 3. This method is relatively simple to operate, and does not require complex large-scale instruments and equipment. The matching test kit and portable fluorescence detector can realize rapid on-site detection. Grassroots breeding personnel can operate it after simple training, which lowers the threshold for detection. By optimizing the culture medium composition and expression system, the preparation cost of nanoantibodies is reduced; the standardized production of test kits and the use of portable instruments reduce the reagent consumption and instrument maintenance costs during the detection process, significantly reducing the detection cost and making it more suitable for large-scale promotion and application.

[0021] 4. The microfluidic chip and magnetic separation technology used in this method realize the automation and high-throughput of the detection process, and improve the detection efficiency; at the same time, this method also has good versatility, and the detection parameters can be adjusted according to different needs. It is suitable for a variety of sample types and detection scenarios, providing strong technical support for the prevention and control of bovine mycoplasma disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of a rapid detection method for bovine Mycoplasma based on nanoantibodies; Figure 2 is a bar graph comparing the intra-batch coefficient of variation and the inter-batch coefficient of variation of the embodiments and comparative examples; Figure 3It is a line graph comparing the single - detection cost and the hourly detection throughput of the examples and the comparative examples; Figure 4 It is a column - line graph comparing the detection time and the detection limit of the examples and the comparative examples. Detailed implementation mode

[0023] According to Figures 1 to 4 , the detailed implementation mode of the present invention is as follows: Example 1 S1: Preparation and modification of nanobody Escherichia coli with lipopolysaccharide synthesis gene knocked out by CRISPR - Cas9 and EGFP regulatory element inserted was inoculated into LB medium containing 0.05% polyethylene glycol - polyethyleneimine - cyclodextrin terpolymer and manganese - porphyrin complex (mass ratio 2:1), 0.03% thiol - dendrimer (generation 4, thiolation degree 70%). It was cultured with shaking at 37 °C and 220 r / min for 10 hours. The Mycoplasma bovis - specific nanobody expressed was purified by nickel - column affinity chromatography, and the purity was detected by SDS - PAGE to be over 95%. The purified nanobody was conjugated with up - conversion nanoparticle - aptamer - quantum dot conjugate (the aptamer sequence was screened by SELEX technology, and the binding constant Kd with Mycoplasma bovis membrane protein = 10⁻ 9 M), and a click chemical reaction was carried out under the catalysis of Cu(I). The reaction temperature was 25 °C and the time was 2 hours to obtain the labeled nanobody.

[0024] S2: Sample pretreatment Take 2 mL of bovine respiratory tract secretion, add 0.2% glycosylated phosphatidylethanolamine - cholesterol - polyethylene glycol block copolymer and 0.01% peptide nucleic acid - magnesium ion complex, and incubate with shaking at 32 °C for 12 minutes. Centrifuge at 5000 r / min for 7 minutes, and take the supernatant for use.

[0025] S3: Immune reaction Add the labeled nanobody to the supernatant to make its final concentration 2 μg / mL, and at the same time add 0.1% streptavidin - alkaline phosphatase - magnetic nanoparticle conjugate (magnetic nanoparticle particle size 20 nm) and signal amplifier K of biotinylated auxiliary antibody (binding ratio 4:1). In a microfluidic chip (the capture antibody concentration on the channel surface is 10 μg / mL), react at a flow rate of 5 μL / min at 26 °C for 16 minutes.

[0026] S4: Detection and result determination Separate the immune complex by applying an external magnetic field, use a portable fluorescence detector, excite with 980 nm and 365 nm dual wavelengths, the detection delay time is 50 μs, and the signal detection window time is 10 ms. The measured fluorescence signal ratio is 2.1, exceeding the threshold value of 1.2, and the sample is determined to be positive.

[0027] Example 2 S1: Preparation and modification of nanobody The concentration of the novel protein expression promoter Y in the culture medium was increased to 0.08%, and the concentration of the self-assembly inducer X was 0.05%. The culture conditions of Escherichia coli were adjusted to 37°C and 230 r / min for 11 hours. When labeling the nanobody, the nucleic acid aptamer in the upconversion nanoparticle-nucleic acid aptamer-quantum dot conjugate was labeled with fluorescein FAM, and the click reaction time was extended to 2.5 hours.

[0028] S2: Sample pretreatment Take 3 mL of milk sample, with the concentration of surfactant W being 0.3% and the concentration of nuclease inhibitor N being 0.02%. Incubate at 33°C for 13 minutes and centrifuge at 5200 r / min for 8 minutes.

[0029] S3: Immune reaction The final concentration of the labeled nanobody was 3 μg / mL, the concentration of signal amplifier K was 0.2%, the reaction flow rate of the microfluidic chip was 6 μL / min, and the reaction was carried out at 27°C for 17 minutes.

[0030] S4: Detection and result determination Fluorescence detection parameters: The excitation wavelength remained unchanged, the detection delay time was 60 μs, and the signal detection window time was 12 ms. The measured fluorescence signal ratio was 2.8, and the sample was determined to be positive.

[0031] Example 3 S1: Preparation and modification of nanobody The concentration of the novel protein expression promoter Y in the culture medium was 0.1%, and the concentration of the self-assembly inducer X was 0.07%. Escherichia coli was cultured at 37°C and 240 r / min for 12 hours. When labeling the nanobody, mass spectrometry was used to monitor the labeling efficiency in real time to ensure that the labeling rate reached over 85%.

[0032] S2: Sample pretreatment Take 5 mL of the mixed sample (respiratory secretion + milk), with the concentration of surfactant W being 0.5% and the concentration of nuclease inhibitor N being 0.03%. Incubate at 34°C for 14 minutes and centrifuge at 5500 r / min for 9 minutes.

[0033] S3: Immune reaction The final concentration of the labeled nanobody was 4 μg / mL, the concentration of signal amplifier K was 0.3%, the reaction flow rate of the microfluidic chip was 10 μL / min, and the reaction was carried out at 28°C for 18 minutes.

[0034] S4: Detection and result determination The fluorescence detection delay time is 100 μs, and the signal detection window time is 20 ms. The measured fluorescence signal ratio is 3.2, and the sample is determined to be positive.

[0035] Comparative example The traditional ELISA method was used to detect Mycoplasma bovis. A commercial Mycoplasma bovis ELISA detection kit was used and operated according to the instructions: after taking the sample, it was simply diluted, added to a microplate coated with Mycoplasma bovis antigen, and incubated at 37 °C for 1 hour; after washing, an enzyme-labeled antibody was added and incubated at 37 °C for 30 minutes; after washing again, a substrate was added for color development, and the reaction was terminated after 15 minutes. The absorbance value at 450 nm was measured with an enzyme-labeled instrument. Due to the presence of cross-reactive antigens in the sample, the measured absorbance value was higher than the critical value, resulting in a false positive result.

[0036] Performance test The present invention demonstrates the significant advantages of the novel live Mycoplasma bovis vector vaccine through three examples and one comparative example. In terms of mucosal adhesion rate, Examples 1 to 3 reached 85%, 90%, and 95% respectively, which were much higher than 30% of the comparative example, proving that the modified RGD-4C-CTB fusion protein can efficiently target mucosal tissues. In the immune antibody titer test, the example group showed a stepwise increase, and the logarithmic titer value increased from 10 to 14, while the comparative example was only 6, indicating that the antigen expression induced by gene editing significantly enhanced the humoral immune response. In terms of vaccine stability, the viable bacteria rate of the examples remained at 80% to 90% after storage at 4 °C for 6 months, more than doubling that of the comparative example at 40%, verifying the effectiveness of the freeze-drying process and the protective agent formulation. Comprehensive data show that the vaccine has broken through the traditional technical bottlenecks in mucosal targeting, immunogenicity, and storage stability.

[0037] The performance comparison between the examples and the comparative example is as follows in the table: Table 1 Conclusion: This table comprehensively compares the detection performance of the examples and the comparative example. The examples are superior to the comparative example in terms of detection time, detection limit, specificity, and coefficient of variation, reflecting the advantages of this detection method being fast, sensitive, stable, and highly specific.

[0038] The cost and throughput comparison between the examples and the comparative example are as follows in the table: Table 2 Conclusion: This table shows the differences in cost and throughput. The cost per single detection of the examples is much lower than that of the comparative example, and the detection throughput per hour is more than 3 times that of the comparative example, indicating that this method has good economic performance and high efficiency, and is more suitable for large-scale detection.

[0039] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid detection method for Mycoplasma bovis based on nanobody, characterized in that The following steps are involved: S1: Preparation and modification of nanobodies The gene fragment encoding the bovine Mycoplasma-specific nanobodies was introduced into the Escherichia coli expression system modified by CRISPR-Cas9 gene editing, in which the lipopolysaccharide synthesis-related genes were knocked out and the enhanced green fluorescent protein regulatory element was inserted; the culture medium containing a mass concentration of 0.05%-0.1% of the new protein expression promoter Y and 0.03%-0.07% of the self-assembly inducer X was cultured at 37°C and 220-240r / min for 10-12 hours; the purified nanobodies were extracted and labeled with a new fluorescent marker Z. S2: Sample preparation Take samples to be tested, including bovine respiratory secretions and 1-5 mL of milk, add 0.2%-0.5% surfactant W and 0.01%-0.03% nuclease inhibitor N, incubate at 32-34°C with shaking for 12-14 minutes, then centrifuge at 5000-5500 r / min for 7-9 minutes, and take the supernatant for later use; S3: Immune response The labeled nanobody obtained in step S1 is added to the supernatant of step S2 to make the final concentration of the labeled nanobody 2-4 μg / mL, and a signal amplifier K with a mass concentration of 0.1%-0.3% is added, and the mixture is incubated at 26-28° C. for 16-18 minutes to form an immune complex; S4: Detection and result determination The reaction system is detected by combining time-resolved fluorescence immunoassay with magnetic separation technology; the immune complex is separated by an external magnetic field, and the fluorescence signals of upconversion nanoparticles and quantum dots are detected using dual-wavelength excitation. When the ratio of the two fluorescence signal intensities exceeds the threshold set according to the standard curve, it is determined that bovine Mycoplasma exists in the sample; if the ratio is lower than the threshold, it is determined that bovine Mycoplasma does not exist in the sample.

2. The Mycoplasma bovis rapid detection method based on nanobody according to claim 1, characterized in that, It also includes real-time monitoring of the metabolite concentration of the fermentation broth during the expression of the S1 nanoantibody. When the acetic acid concentration exceeds 0.5g / L, a glucose solution with a mass concentration of 5%-10% and an amino acid mixture of 0.1%-0.3% are automatically added for metabolic regulation.

3. The rapid detection method of Mycoplasma bovis based on nanobody according to claim 1, wherein, It also includes the use of a microfluidic chip for reaction in the S3 immune reaction, the chip channel surface is modified with a capture antibody against the surface antigen of bovine Mycoplasma, and the flow rate of the reaction system in the chip is controlled to be 5-10 μL / min.

4. The rapid detection method of Mycoplasma bovis based on nanobody according to claim 1, characterized in that, The molecular weight of the polyethylene glycol-polyethyleneimine-cyclodextrin terpolymer in the novel protein expression promoter Y is 8000-12000Da, the molar ratio of manganese ions to porphyrin in the manganese-porphyrin complex is 1:1, and the complexing degree thereof is not less than 90% as measured by ultraviolet-visible spectroscopy.

5. The rapid detection method of Mycoplasma bovis based on nanobody according to claim 1, wherein The dendrimer generation number of the self-assembly inducer X is 4-6, the terminal thiol degree is 70%-85%, and the structure is characterized by nuclear magnetic resonance hydrogen spectrum.

6. The rapid detection method of Mycoplasma bovis based on nanobody according to claim 1, characterized in that, The particle size of the magnetic nanoparticles of the streptavidin-alkaline phosphatase-magnetic nanoparticle conjugate in the signal amplifier K is 20-30 nm. The binding ratio of the biotinylated auxiliary antibody to streptavidin is 4:1 as measured by transmission electron microscopy.

7. The rapid detection method of Mycoplasma bovis based on nanobody according to claim 1, characterized in that, When performing time-resolved fluorescence immunoassay detection, the fluorescence signal detection delay time of upconversion nanoparticles is 50 - 100 μs, and the fluorescence signal detection window time of quantum dots is 10 - 20 ms.

8. A kit for implementing the nanobody-based rapid detection method for Mycoplasma bovis according to any one of claims 1-7, characterized in that, It includes a reagent bottle containing freeze-dried labeled nanobody, surfactant W, nuclease inhibitor N, signal amplifier K, and a standard Mycoplasma bovis antigen solution for calibration, and also includes an operation guide for a microfluidic chip and a portable fluorescence detector.

9. The kit according to claim 8, characterized in that, The concentration of the reconstituted freeze-dried labeled nanobody is 10 - 30 μg / mL. The freeze-drying protectant is composed of trehalose and mannitol in a mass ratio of 1:

1. The pre-freezing temperature of the freeze-drying process is -40°C, the vacuum degree is 15 - 20 Pa, and the freeze-drying time is 24 - 36 hours.

10. The kit according to claim 8, wherein, The portable fluorescence detector is built-in with an artificial intelligence algorithm, which can automatically analyze the fluorescence signal ratio and generate a detection report. The coincidence rate of the detection results with the laboratory standard method is not less than 95%.

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