Method for quantitatively detecting viable count of mycoplasma bovis

Abstract

The invention belongs to the technical field of quantitative detection of microorganisms, and particularly relates to a method for quantitatively detecting the viable count of mycoplasma bovis. The method for quantitatively detecting the viable count of the mycoplasma bovis is finally obtained by exploring the linear relation between the protein concentration and the viable count of multiple batches of mycoplasma bovis liquid samples after optimization of experimental conditions such as bacterium liquid preservation conditions, ultrasonic crushing modes, crushing duration and sample treatment modes, and the method is good in stability, high in timeliness and high in accuracy. The detection result is accurate, and a convenient and effective method is provided for rapid detection of the viable count of the mycoplasma bovis liquid.

Description

Technical Field

[0001] This invention belongs to the field of quantitative detection technology of microorganisms, specifically relating to a method for quantitatively detecting the number of viable Mycoplasma bovis. Background Technology

[0002] Current methods for counting viable bovine mycoplasma use the plate count method. This method involves diluting the sample and spreading it on a solid culture medium, with each viable bacterium forming a visible colony (CFU, colony-forming unit). The specific procedure involves serially diluting the sample (usually 10-fold), spreading or pouring an appropriate amount of the dilution onto plates, incubating under suitable conditions, and then counting the colonies. While this method has the advantage of directly reflecting the number of viable bacteria, it also has disadvantages such as being time-consuming (24-48 hours), the counting results being affected by human counting errors, and the limitation to counting only culturable microorganisms. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for quantitative detection of viable bovine mycoplasma bacteria, addressing the shortcomings of the prior art.

[0004] To address the aforementioned technical problems, this invention discloses a method for quantitatively detecting the number of viable bovine mycoplasma bacteria. The specific technical solution is as follows: This invention provides a method for quantitatively detecting the number of viable bovine mycoplasma bacteria, comprising the following steps: The bovine mycoplasma bacterial suspension was centrifuged and resuspended to obtain a resuspended bacterial solution. Protein concentration was measured, and the bovine mycoplasma viable count was calculated based on a fitted standard curve of protein concentration and bovine mycoplasma viable count. This method is not a disease diagnostic method.

[0005] The bovine mycoplasma bacterial solution is a bacterial solution stored at 2-8 ℃ for 0-24 h.

[0006] The protein concentration of the resuspended bacterial solution was determined according to either method (1) or (2) below: (1) Directly detect the protein concentration of the resuspended bacterial solution; (2) After the resuspended bacterial solution is broken, it is centrifuged again, and the supernatant is taken to detect the protein concentration.

[0007] The aforementioned fragmentation is non-contact focused ultrasonic fragmentation. The duration of the non-contact focused ultrasonic fragmentation is 50-60 minutes. In some embodiments of the present invention, the ultrasonic fragmentation procedure consists of 15-25 seconds of ultrasonic treatment followed by a 10-15 second pause.

[0008] The second centrifugation is described as centrifugation at 10000~12000 r / min for 10~20 min. In some embodiments of the present invention, the second centrifugation is described as centrifugation at 12000 r / min for 10 min.

[0009] The protein concentration was detected using a BCA protein quantification kit.

[0010] When the protein concentration is obtained by directly detecting the resuspended bacterial solution, the fitting standard curve is shown in Equation I: y = 0.0106*x-0.4478 (I), Where x is the protein concentration obtained by direct detection, in μg / mL; y is the viable cell count, in 10⁻¹⁰. 9 CFU / mL.

[0011] Wherein, when the protein concentration is obtained by centrifuging the resuspended bacterial solution again after disruption and then detecting the supernatant, the fitting standard curve is as shown in Equation II: y=0.111*x1-0.2967 (II) Where x1 is the protein concentration obtained by centrifugation again after disruption, measured in μg / mL; y is the viable cell count, 10-1. 9 CFU / mL.

[0012] Wherein, if the resuspension of Mycoplasma bovis prepared from the bacterial culture has been concentrated or diluted before protein concentration detection, then the fitting standard curve is obtained by multiplying either Formula I or Formula II by the dilution factor (or the reciprocal of the concentration factor), specifically: When the protein concentration is obtained by directly detecting the resuspended bacterial solution after concentration or dilution, the fitting standard curve is shown in Equation III: y = (0.0106*x - 0.4478)*n (III), When the protein concentration is obtained by centrifuging the resuspended bacterial culture after concentration or dilution and then testing the supernatant, the fitting standard curve is shown in Equation IV: y=(0.111*x1-0.2967)*n (IV).

[0013] In Formulas III and IV, n is the reciprocal of the dilution factor or concentration factor. For example, if the concentration is 4 times, n is 0.25; if the concentration is 4 times, n is 4, which means the original concentration is restored.

[0014] The fitting standard curve between protein concentration and viable count of *Mycoplasma bovis* was obtained as follows: After centrifugation and resuspending, the *Mycoplasma bovis* bacterial solution was concentrated or diluted at different ratios by adjusting the resuspending volume. Protein concentration was detected using the BCA method and viable count was detected using the plate count method. Based on the protein concentration and actual viable count of multiple batches of *Mycoplasma bovis* solution after different concentration or dilution ratios and under each treatment method, the fitting standard curve between protein concentration and viable count was calculated.

[0015] The bovine mycoplasma bacterial solution mentioned herein includes a bacterial solution obtained through fermentation culture of bovine mycoplasma. In other embodiments of the present invention, the small-scale culture bacterial solution includes shake flask culture solution and / or reactor culture solution.

[0016] The resuspension is performed using PBS buffer. In some embodiments of the present invention, the pH of the PBS buffer is 7.2-7.4, and the ion concentration is 0.1M. In some embodiments of the present invention, the volume of the PBS buffer used for resuspension is 0.25 to 4 times the volume of the Mycoplasma bovis culture. In other embodiments of the present invention, the volume of the PBS buffer used is 0.25 times the volume of the Mycoplasma bovis culture, that is, the Mycoplasma bovis culture is concentrated 4 times.

[0017] During the research and development process, the following aspects of this technical solution were adjusted and optimized: determination of the preservation conditions before detection of *Bacillus cereus* fluid; determination of the optimal ultrasonic disruption time for *Bacillus cereus* fluid; linear relationship between the protein concentration after disruption and the protein concentration without disruption and the actual viable count, and intra-batch repeatability verification; comparison of the effect of protein concentration after disruption and without disruption on the viable count detection results; optimal pretreatment method for protein detection of *Bacillus cereus* fluid samples; applicability of different fitting standard curves to different types of *Bacillus cereus* samples; and verification of the stability of the detection method.

[0018] Through the above adjustments and optimizations, the optimal storage conditions for *Bacillus cereus* solution before testing were determined to be 2–8°C for 24 hours. By exploring the optimal ultrasonic duration for concentrated *Bacillus cereus* solution, the optimal ultrasonic duration for a 4x concentrated *Bacillus cereus* solution sample was found to be 60 seconds. The method is compatible with other low-concentration *Bacillus subtilis* slurries, ensuring the stability and accuracy of the experimental method. By exploring the linear relationship between the protein concentration after disruption and the actual viable count of *Bacillus subtilis* slurry, it was found that the linear relationship between these two concentrations is good and has stable intra-batch repeatability. Standard curves were established using data from more than 10 batches, and standard curve fitting was completed for the protein concentration after disruption and the viable count of *Bacillus subtilis* slurry. By comparing the relative errors between the calculated viable count and the actual viable count using the standard curve for *Bacillus subtilis* slurry samples, it was found that the relative error between the calculated and actual viable count of the sample using the standard curve for viable count with undisrupted protein concentration is smaller than that using the standard curve for viable count with protein concentration after disruption. Through the determination and optimization of the above experimental conditions, it is possible to achieve a throughput of up to 12 samples (limited by the centrifugation throughput of a 1 mL centrifuge rotor) within 4... The viable count can be rapidly detected within h (the plate count method requires 24~48h), and the relative error between the viable count calculated by the method of this invention and the actual viable count obtained by the plate count method is <10%.

[0019] Beneficial effects: This invention establishes a fitting standard curve between the protein concentration and viable count of Mycoplasma bovis broth. By rapidly detecting the protein concentration of a Mycoplasma bovis broth sample, the viable count of the sample can be calculated. This method greatly improves the timeliness of obtaining the viable count of Mycoplasma bovis broth samples and, to a certain extent, solves the problems of low timeliness, long cycle, and large human error in Mycoplasma bovis viable count.

[0020] Specifically, this invention, through exploratory experiments on the linear relationship between protein concentration and viable cell count in multiple batches of *Bacillus subtilis* broth samples, fitted standard curves for the linear relationship between protein concentration after disruption and protein concentration without disruption and viable cell count. Furthermore, it was found that using the standard curves for protein concentration and viable cell count, the relative error between the calculated viable cell count and the actual viable cell count obtained by the plate count method for samples of different culture types (including shake flask culture, reactor culture, and *Bacillus subtilis* samples cultured in 100L pilot-scale) was <10%. The specific beneficial effects are as follows: (1) The fitting standard curve calculation method described in this invention establishes a fitting standard curve between protein concentration and viable cell count. The viable cell count is calculated by substituting the protein concentration of the sample to be tested into the fitting standard curve. The detection accuracy is high, and the error range with the measured value is within 10%. This application has used multiple batches of samples under various culture conditions to verify the method, and the error can be reasonably controlled. The method of this invention has good stability and repeatability. (2) For testing personnel, the method of the present invention only requires the determination of protein content according to the BCA method, which has low technical requirements and the test results are not affected by human testing errors compared with the plate counting method. (3) The method of the present invention has a faster counting time. The viable count results can be obtained within 4 hours for up to 12 samples, which is more than 6 times shorter than the ordinary plate counting method. Attached Figure Description

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0022] Figure 1 This is a graph showing the linear relationship between protein concentration and viable bacterial count after disruption by an ultrasonic cell disruptor.

[0023] Figure 2 This is a graph showing the linear relationship between protein concentration and viable bacteria count after high-pressure homogenization.

[0024] Figure 3 This is a graph showing the linear relationship between protein concentration and viable bacteria count after being broken up by a non-contact focused ultrasonic breaking device.

[0025] Figure 4 This is a graph showing the changes in protein concentration and ultrasonic duration after ultrasonic disruption by a non-concentrated ultrasonic disruptor.

[0026] Figure 5 This is a graph showing the linear relationship between protein concentration and viable cell count after lysis without centrifugation.

[0027] Figure 6 This is a graph showing the linear relationship between protein concentration and viable bacterial count after centrifugation following disruption.

[0028] Figure 7 The graph shows the linear relationship between protein concentration and viable cell count in the first batch of *Bacillus subtilis* broth. In the graph, A represents the first batch after disruption and repeat 1, B represents the first batch after disruption and repeat 2, C represents the first batch after disruption and repeat 3, D represents the first batch without disruption and repeat 1, E represents the first batch without disruption and repeat 2, and F represents the first batch without disruption and repeat 3.

[0029] Figure 8 The graph shows the linear relationship between protein concentration and viable cell count in the second batch of *Bacillus cereus* broth. In the graph, A represents the second batch after disruption and repeat 1; B represents the second batch after disruption and repeat 2; C represents the second batch after disruption and repeat 3; D represents the second batch without disruption and repeat 1; E represents the second batch without disruption and repeat 2; and F represents the second batch without disruption and repeat 3.

[0030] Figure 9 This is a fitted curve of protein concentration versus viable cell count after centrifugation of multiple batches of *Bacillus subtilis* broth.

[0031] Figure 10 This is a fitted curve of the concentration of unbroken protein and the number of viable bacteria in multiple batches of *Bacillus subtilis* culture. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0033] In the following examples, the PBS has a pH of 7.2-7.4 and an ion concentration of 0.1M.

[0034] In the following examples, the Pierce BCA protein assay kit (purchased from Thermo Fisher, catalog number: 23227) was used to perform quantitative detection of protein concentration in the resuspended *Bacillus subtilis* culture after centrifugation and medium exchange.

[0035] The bovine mycoplasma strain used in the following examples is the bovine mycoplasma HM strain, which was isolated, identified and preserved by Tiankang Biopharmaceutical Co., Ltd.

[0036] Example 1: Sample preparation method and standard curve preparation method for Mycoplasma bovis suspension. The *Bacillus subtilis* culture used in this embodiment was obtained by culturing on Thiaucourt's medium. The cultured bacterial solution with the medium was used as the original volume solution, and then concentrated or diluted with PBS to obtain bacterial solutions of different multiples. The specific processing methods are as follows: 1. Treatment with 4x concentration of bacterial culture (4XNS) (1) Mix the original bacterial culture thoroughly. Use a 1 mL pipette to accurately measure 4 1 mL original bacterial cultures into 1.5 mL centrifuge tubes and name them the first, second, third and fourth centrifuge tubes respectively. Centrifuge at 12000 r / min for 30 min. Carefully remove the 4 centrifuge tubes to avoid splashing the supernatant (culture medium) onto the centrifuge tube cap. Use a pipette to discard the supernatant after centrifugation twice. Use a pipette to remove most of the supernatant the first time. After standing for 3-5 min, use a pipette to remove the small amount of supernatant that has accumulated at the bottom. You will get 4 centrifuge tubes containing bacterial precipitate for subsequent concentration. (2) Add 500 μL of PBS to the first centrifuge tube and shake thoroughly to dissolve the precipitate completely. After short-term centrifugation, resuspend the precipitate in the first centrifuge tube and transfer it to the second centrifuge tube. Add 500 μL of PBS to the first centrifuge tube and shake thoroughly to dissolve the precipitate. Resuspend the precipitate in the tube a second time to ensure that the precipitate in the tube is resuspended in PBS. Short-term centrifugation is then used for later use. (3) Shake the second centrifuge tube obtained in step (2) thoroughly to dissolve the precipitate completely. After a short time, resuspend the precipitate in the second centrifuge tube and transfer it to the third centrifuge tube. Then add 500 μL of the second resuspension solution obtained in step (2) to the second centrifuge tube, shake thoroughly to mix, and then aspirate for later use. The first centrifuge tube becomes an empty tube and is discarded. (4) Shake the third centrifuge tube obtained in step (3) thoroughly to dissolve the precipitate completely. After a short time, resuspend the precipitate in the third centrifuge tube and transfer it to the fourth centrifuge tube. Then add 500 μL of the second resuspended liquid from the second centrifuge tube to the third centrifuge tube, shake thoroughly to mix, and then aspirate for later use. The second centrifuge tube will become an empty tube and should be discarded. (5) Shake the fourth centrifuge tube obtained in step (4) thoroughly to dissolve the precipitate completely. Then combine the 500 μL of the secondary resuspension from the third centrifuge tube with the fourth centrifuge tube. The third centrifuge tube will become an empty tube and should be discarded. (6) Shake thoroughly to mix the fourth centrifuge tube obtained in step (5) (combine the 4 tubes of precipitate into this tube), then centrifuge briefly to obtain a 4-fold concentrated bacterial solution for later use.

[0037] 2. Treatment with 2x concentration of bacterial culture (2XNS) (1) Mix the original bacterial culture thoroughly. Use a 1 mL pipette to accurately measure two 1 mL original bacterial cultures into 1.5 mL centrifuge tubes, which are named the first and second centrifuge tubes respectively. After centrifuging at 12000 r / min for 30 min, carefully remove the two centrifuge tubes to avoid splashing the supernatant (culture medium) onto the centrifuge tube cap. Use a pipette to discard the supernatant after centrifugation twice. Use a pipette to remove most of the supernatant the first time. After standing for 3-5 min, use a pipette to remove the small amount of supernatant that has accumulated at the bottom, and obtain two centrifuge tubes containing bacterial precipitate for subsequent concentration processing. (2) Add 500 μL of PBS to the first centrifuge tube and shake thoroughly to dissolve the precipitate completely. After short-term centrifugation, resuspend the precipitate in the first centrifuge tube and transfer it to the second centrifuge tube. Add 500 μL of PBS to the first centrifuge tube and shake thoroughly to dissolve the precipitate. Resuspend the precipitate in the tube a second time to ensure that the precipitate in the tube is resuspended in PBS. After short-term centrifugation, combine the precipitate and add it to the second centrifuge tube. The first centrifuge tube becomes an empty tube and is discarded. (3) Shake thoroughly to mix the second centrifuge tube obtained in step (2) (combine the two tubes of precipitate into this tube), then centrifuge briefly to obtain a 2-fold concentrated bacterial solution for later use.

[0038] 3. Treatment with original double-distilled bacterial culture (1X) Mix the original culture medium thoroughly, accurately measure 1 mL of the original culture medium using a 1 mL pipette, centrifuge at 12000 r / min for 30 min, discard the culture medium supernatant, add 1 mL of PBS and vortex to resuspend. The resuspended culture medium of the same volume is the original culture medium replacement culture medium.

[0039] 4. Treatment with a 2-fold dilution of the bacterial solution (2XXS) Thoroughly mix the original bacterial culture, accurately measure 500 μL of the original bacterial culture sample into a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 30 min, carefully remove the centrifuge tube, avoiding splashing the supernatant (culture medium) onto the centrifuge tube cap, and discard the supernatant twice using a pipette. For the first centrifugation, use a 1 mL pipette to remove most of the supernatant, let it stand for 3-5 min, and then use a 100 or 200 μL pipette to remove the small amount of supernatant that has accumulated at the bottom; add 1 mL of PBS to the centrifuge tube and vortex thoroughly to completely dissolve the precipitate, then centrifuge briefly to obtain a 2-fold diluted bacterial culture.

[0040] 5. Treatment with a 4-fold dilution of the bacterial culture (4XXS) Thoroughly mix the original bacterial culture, accurately measure 250 μL of the bacterial culture sample into a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 30 min, carefully remove the centrifuge tube, avoiding splashing the supernatant (culture medium) onto the centrifuge tube cap, and discard the supernatant twice using a pipette. For the first centrifugation, use a 1 mL pipette to remove most of the supernatant, let it stand for 3-5 min, and then use a 100 or 200 μL pipette to remove the small amount of supernatant that has accumulated at the bottom; add 1 mL of PBS to the centrifuge tube and vortex thoroughly to completely dissolve the precipitate, then centrifuge briefly to obtain a 4-fold diluted bacterial culture.

[0041] Five bacterial suspension concentrations (4x concentrated NS, 2x concentrated NS, original 1x replacement bacterial suspension, 2x diluted 2XXS, and 4x diluted 4XXS) were used to establish the standard curve.

[0042] Example 2: Determination of Preservation Conditions for Bovine Mycoplasma Fluid Because the cultured Mycoplasma bovis suspension samples are stored in the culture medium before testing, prolonged storage may lead to bacterial growth or death, affecting the accuracy of the test results and impacting both the establishment of the standard curve and the sample detection results. Therefore, this embodiment optimized the storage temperature (2~8 ℃, -20 ℃) ​​and storage time (0~24 h, 48 h) of the Mycoplasma bovis suspension. Protein concentration and viable cell count were measured under different storage conditions to determine the optimal storage temperature and duration, laying the foundation for the establishment of the standard curve and the accuracy of the sample detection results. The validation results of storing the Mycoplasma bovis suspension at 2~8 ℃ and -20 ℃ for different times are shown in Tables 1 and 2. The storage conditions described refer to the storage of the original concentration of Mycoplasma bovis suspension. After storage, the original concentration suspension was further concentrated or diluted, and the viable cell count or protein concentration was determined. The protein concentration determination method described in this embodiment involves concentrating or diluting the preserved bovine mycoplasma bacterial solution according to the method described in Example 1, followed by breaking the solution using a non-contact focused ultrasonic disruptor. The disruption conditions are: ultrasonic time 40 min, ultrasonic power 100%, ultrasonic 20 s interval followed by a 15 s pause. The protein concentration is then measured after ultrasonic disruption. The viable cell count determination method involves concentrating or diluting the preserved bovine mycoplasma bacterial solution according to the method described in Example 1, and then directly measuring the viable cell count of the treated solution.

[0043] Table 1. Changes in protein concentration (μg / mL) of *Corynebacterium bovis* culture after disruption under different storage conditions.

[0044] Table 2. Viable bacterial counts of *Bacillus subtilis* culture under different storage conditions (x10⁻¹⁰) 9 Table of changes in CFU / mL

[0045] The validation results of storing *Bacillus subtilis* culture at 2–8℃ and -20℃ for 48 h showed that after treatment with five concentration gradients, the coefficient of variation (CV) of protein concentration and the coefficient of variation (CV) of viable cell count of *Bacillus subtilis* culture stored at 2–8℃ for 48 h were <10% and <15%, respectively. However, the CV values ​​of protein concentration and viable cell count stored at -20℃ for 48 h were significantly higher than those stored at 2–8℃. Therefore, *Bacillus subtilis* culture samples were stored at 2–8℃, and the stability of protein concentration and viable cell count was validated after 24 h of storage at 2–8℃. The validation results are shown in Table 3.

[0046] Table 3. Changes in protein concentration and viable cell count of *Bacillus subtilis* resuspended in PBS after storage at 2–8 °C for 24 h.

[0047] The CV values ​​of protein concentration and viable cell count changes of *Bacillus subtilis* culture after storage at 2-8℃ for 0h, 6h, 10h, and 24h were all <15%, indicating that the detection of the sample culture was completed within 24h at 2-8℃, which can ensure the stability of the sample and improve the accuracy of the method detection results to a certain extent.

[0048] Example 3: Determination of the disruption method for bovine mycoplasma bacterial suspension This embodiment optimized the disruption method for freshly cultured *Bacillus subtilis* culture stored at 2-8℃ for 24 hours, screening ultrasonic cell disruptors, high-pressure homogenizers, and non-contact focused ultrasonic disruptors. Details are as follows: 1. Ultrasonic cell disruptor method: Following the method described in Example 1 (all volumes need to be proportionally increased to a post-treatment bacterial solution volume > 2 mL), prepare 4x concentrated bacterial solution, 2x concentrated bacterial solution, original solution with replacement solution, 2x diluted bacterial solution, and 4x diluted bacterial solution. Take 400 μL of each solution in a biosafety cabinet for viable cell counting. Next, take 2 mL of each concentration of *Ispermia ulmoides* solution and disrupt each concentration individually using an ultrasonic cell disruptor (Ningbo Xinzhi, JY96-II; amplitude transformer φ2mm). The disruption conditions are: ultrasonic power 30%, 5s sonication followed by 5s pause, with a total sonication time of 20 min for each sample. Quantitative protein concentration is measured for each concentration gradient of disrupted bacterial solutions, and a linear relationship graph between protein concentration and viable cell count is plotted against the actual viable cell count results, as shown below. Figure 1 As shown.

[0049] 2. Crushing method using a high-pressure homogenizing crusher: Following the method described in Example 1 (all volumes need to be proportionally scaled up to a post-treatment bacterial solution volume > 82 mL), prepare 4x concentrated bacterial solutions, 2x concentrated bacterial solutions, original-volume replacement bacterial solutions, 2x diluted bacterial solutions, and 4x diluted bacterial solutions. Take 400 μL of each solution in a biosafety cabinet for viable count testing. Then, take 80 mL of each concentration of resuspended bacterial solution and homogenize each concentration of *Ispermia ulmoides* solution using a high-pressure homogenizer. The homogenization conditions are: high pressure 1280 bar, low pressure 18-20 bar, and homogenization times twice. Quantitative protein concentration is measured in the homogenized bacterial solutions of each concentration gradient, and a linear relationship graph between protein concentration and viable cell count is plotted against the actual viable cell count results, as shown below. Figure 2 As shown.

[0050] 3. Non-contact focused ultrasonic breaking method: Following the method described in Example 1, 4x concentrated bacterial suspension, 2x concentrated bacterial suspension, original-volume replacement bacterial suspension, 2x diluted bacterial suspension, and 4x diluted bacterial suspension were prepared. 400 μL of each was taken from each suspension and sent for viable cell counting in a biosafety cabinet. Then, 100 μL was placed in a 1.5 mL EP tube and briefly removed. Using a pre-cooled non-contact focused ultrasonic disruptor, each concentration of *Ischemicum bovis* suspension was simultaneously placed on a 2 mL EP tube adapter for ultrasonic disruption. The disruption conditions were: ultrasonic time 40 min, ultrasonic power 100%, ultrasonic 20 s, pause 15 s. Protein concentration was quantitatively measured in the disrupted bacterial suspensions at each concentration gradient, and a linear relationship graph between protein concentration and viable cell count was plotted. Figure 3 As shown.

[0051] The above three disruption methods were used to disrupt three batches of *Bacillus subtilis* culture. The protein concentration and viable cell count results are shown in Table 4. Figures 1-3 The results in Table 4 indicate that the non-contact focused ultrasonic breaking device exhibits the best linear relationship between protein concentration and viable cell count in the *Bacillus subtilis* broth after fragmentation. The linearity R0 for protein concentration and viable cell count was highest in all three batches of broth. 2 All values ​​were >0.99. The protein concentration after being broken down using a high-pressure homogenizer was similar to that of a non-contact focused ultrasonic disruptor. However, the non-contact focused ultrasonic disruptor has the advantages of smaller sample volume and higher throughput compared to the other two methods. Therefore, the non-contact focused ultrasonic disruptor was selected for breaking down the Botrytis cinerea bronchiseptica solution.

[0052] Table 4. Results of protein concentration and viable bacterial count after different disruption methods.

[0053] Example 4: Optimal ultrasound duration for *Bacillus subtilis* culture To ensure complete disruption of the bacterial suspension and the accuracy of the standard curve and sample test results, this embodiment further optimizes the ultrasonic duration of the non-contact focused ultrasonic disruptor, building upon Example 3. Since the bacterial suspension needs to be concentrated fourfold during the preparation of the bovine mycoplasma vaccine, this embodiment uses a laboratory-concentrated bovine mycoplasma suspension as the sample.

[0054] Specifically, 100 μL of a 4-fold concentrated bacterial culture was placed in a 1.5 mL EP tube and disrupted using a non-concentrated ultrasonic disruptor. The ultrasonic parameters were the same as in Example 3, except that the ultrasonic time was set to 15–125 min (with 5-min intervals). Protein concentration was measured in all disrupted samples. The results are shown in Table 5. Figure 4 As shown.

[0055] Table 5. Experimental results exploring the optimal ultrasonic duration for 4x concentrated bacterial culture.

[0056] From Table 5 and Figure 4 The results showed that the CV value of the protein concentration after 50 minutes of sonication was <1%, and the protein concentration no longer increased with the increase of sonication time, indicating that the *Bacillus subtilis* broth could be completely broken down after 50 minutes or more. In this example, a sonication time of 60 minutes was selected as the optimal sonication time for the *Bacillus subtilis* broth.

[0057] Example 5: Screening of treatment methods before protein quantification detection after disruption of Botrytis cinerea culture. This embodiment screened the pretreatment method for protein quantification detection after disruption of *Bacillus cereus* culture. Using the storage and ultrasonic disruption conditions determined in the above embodiments, multiple batches of *Bacillus cereus* culture were disrupted and centrifuged or not centrifuged to detect protein concentration and viable cell count, and linear relationship fitting was performed on the detection results of each batch of culture. Specifically, qualified *Bacillus cereus* culture freshly cultured in the laboratory and stored at 2-8 ℃ for 24 h were prepared into 4-fold concentrated (4XNS), 2-fold concentrated (2XNS), original-fold replacement (1X), 2-fold dilution (2XXS), and 4-fold dilution (4XXS) culture according to the method described in Example 1. 400 μL of each culture was taken in a biosafety cabinet and plate viable cell count was performed first.

[0058] No centrifugation after disruption: Take bacterial solutions of various concentration gradients and sonicate them according to the optimized method of the non-contact focused ultrasonic breaking instrument in Example 4. Mix the ultrasonically obtained samples thoroughly and directly perform quantitative detection of protein concentration. Calculate the protein concentration value of each sample and make a linear relationship graph between protein concentration and viable bacteria count. Centrifugation after disruption: Bacterial solutions of various concentration gradients were taken and ultrasonically disrupted according to the optimized method of the non-contact focused ultrasonic disruptor in Example 4. The disrupted samples were centrifuged at 12000 r / min for 10 min. The supernatant was collected for quantitative protein concentration detection. The protein concentration values ​​of each sample were calculated, and a linear relationship graph between protein concentration and viable cell count was plotted. The results are shown in Table 6 and... Figure 5 , Figure 6 As shown.

[0059] Table 6. Comparison of protein concentrations under different pretreatment methods for *Bacillus subtilis* broth.

[0060] Table 6 shows that after treatment with five concentration gradients, the protein concentration of the three batches of *Bacillus subtilis* broth without centrifugation after disruption was higher than that after centrifugation. However, the protein concentration after centrifugation and the viable cell count R were not significantly different. 2 A higher value indicates a better linear relationship, so the broken Botrytis cinerea solution was centrifuged at 12000 r / min for 10 min.

[0061] Example 6: Selection of centrifugation time after disruption of *Bacillus subtilis* culture This embodiment selects the centrifugation time after disruption of the *Bacillus cereus* broth based on Example 5. The same *Bacillus cereus* broth is subjected to ultrasonic disruption followed by centrifugation, with centrifugation times of 5, 10, 15 and 20 min respectively. Five replicates are set for each time. The BCA protein of the centrifuged broth is quantitatively detected, and the results are shown in Table 7.

[0062] Table 7. Protein concentrations (μg / ml) obtained after different centrifugation times of *Bacillus subtilis* culture.

[0063] The experimental results above show that the protein concentration of the *Botrytis cinerea* culture changes the least when centrifuged for 10 minutes, and the protein concentration does not change much when the centrifugation time is extended further. Therefore, 10 minutes is selected as the optimal centrifugation time for this method.

[0064] Example 7: Stability verification of the linear relationship between ruptured and unruptured protein concentration and viable cell count This embodiment verifies the intra- and inter-batch stability of the linear relationship between protein concentration and viable cell count based on the sample processing methods determined in the preceding embodiments and between lysed and centrifuged samples and direct measurement without lysed samples. Three replicate standard curves were performed on two batches of bacterial cultures to verify the stability of the linear relationship. The lysed centrifugation method is the same as in Example 5. The direct measurement without lysed samples method involves mixing bacterial cultures of different concentrations obtained according to the bacterial culture processing method described in Example 1 and then directly measuring the protein concentration. Specific verification results are as follows: 1. The results of the linear relationship and stability verification of the protein concentration and viable cell count of the first batch of *Bacillus subtilis* broth within three replicate tests and the standard curve are shown in Table 8. Figure 7 As shown.

[0065] Table 8. Results of protein concentration and viable cell count in the first batch of *Corynebacterium bovis* liquid, and CV values ​​after three replicates within the same batch.

[0066] 2. The results of the intra-batch triplet detection of protein concentration and viable cell count in the second batch of *Bacillus bovis* broth, and the results of the stability verification of the linear relationship with the standard curve are shown in Table 9. Figure 8 As shown.

[0067] Table 9. Results of protein concentration and viable cell count in the second batch of *Bacillus cereus* liquid, and CV values ​​after three replicates within the same batch.

[0068] From Table 8, Table 9, Figure 7 and Figure 8The results showed that in the intra-batch triple replicate validation experiments of protein concentration and viable cell count for two batches of *Bacillus subtilis* culture, the CV values ​​for protein concentration detection after lysis and centrifugation, and for direct measurement without lysis, were both <10%, and the CV values ​​for viable cell count detection were both <15%. This indicates good intra-batch repeatability and good inter-batch stability of protein concentration and viable cell count. The linear relationships between protein concentration after lysis and centrifugation, and protein concentration measured directly without lysis, and viable cell count showed good linearity both intra-batch and inter-batch for both lysis and lysis conditions. R0 2 The values ​​were all >0.99, indicating that the linear relationship between bovine mycoplasma protein concentration and viable cell count standard curve had good repeatability and stability.

[0069] Example 8: Validation of linearity of multiple batches of Mycobacterium bovis culture and determination of the fitting standard curve Following the experimental method described in Example 7, standard curves were established for protein concentration and viable cell count after lysing and centrifuging 10 batches of *Bacillus subtilis* culture, as well as for direct measurement of protein concentration and viable cell count without lysing. Standard curve fitting was performed on the detection data of protein concentration and viable cell count for all batches under both lysing and unlysing treatments. The detection data results and standard curve fitting results are shown in Tables 10 and 11. Figure 9 , Figure 10 As shown: Table 10. Results of protein concentration and viable cell count after disruption of 10 batches of *Bacillus subtilis* culture.

[0070] Table 11 Results of protein concentration and viable cell count detection in 10 batches of *Bacillus subtilis* culture without disruption.

[0071] According to Table 10, Table 11 and Figure 9 , Figure 10 As shown, regardless of whether the *Bacillus cereus* culture is lysed and centrifuged or directly detected without lysis, the linear relationship R between protein concentration and viable cell count in each batch of culture is as follows: 2 All values ​​were ≥0.99, indicating a good and stable linear relationship between protein concentration and viable cell count in multiple batches of bacterial suspensions under both treatment methods. Based on all the detection data of protein concentration and viable cell count in the above 10 batches of bacterial suspensions, the following results were obtained: The fitting curve of protein concentration and viable cell count after centrifugation after disruption is shown below. Figure 9 As shown, the fitted standard curve is as shown in Equation II: y = 0.0111 * x1 - 0.2967, R 2 =0.95 (II); where x1 is the protein concentration obtained by centrifugation again after disruption, measured in μg / mL; y is the viable cell count, 10 9 CFU / mL.

[0072] Unbroken direct measurement of protein concentration and viable cell count fitting standard curve as follows Figure 10 As shown, the fitted standard curve is as shown in Equation I: y = 0.0106*x - 0.4478, R 2 =0.9505 (I); where x is the protein concentration obtained by direct detection, μg / mL; y is the viable cell count, 10 9 CFU / mL.

[0073] If the bovine mycoplasma bacterial solution has been concentrated or diluted before protein concentration detection, the fitting standard curve is to multiply the above formula I or formula II by the dilution factor (or the reciprocal of the concentration factor). For example, if the concentration is 4 times, multiply by 0.25; if the concentration is 4 times, multiply by 4, that is, restore to the original concentration.

[0074] This embodiment verifies the repeatability and stability of the linear relationship between protein concentration and viable cell count through a linearity verification experiment using 10 batches of *Bacillus subtilis* broth.

[0075] Example 9 Detection of multi-volume culture samples of *Corynebacterium bovis* Based on the fitting curves of multiple batches of *Bacillus cereus* culture in Example 8, the viable counts of *Bacillus cereus* culture samples of different volumes (including shake flasks and 3L reactors) were calculated, and the relative error was calculated with the actual viable count results obtained by plate counting method (CFU method). The relative error calculation results of the fitting curves of protein concentration and viable count after rupture and centrifugation and the fitting curves of protein concentration and viable count without rupture are shown in Tables 12-15.

[0076] Table 12. Calculation of the relative error between the viable cell count calculated from the standard curve fitted after centrifugation and the actual viable cell count.

[0077] Table 13. Calculation of the relative error between the viable cell count calculated from the standard curve fitted after centrifugation and the actual viable cell count (continued).

[0078] Table 14. Calculation of the relative error between the calculated viable count and the actual viable count of the unbroken direct measurement fitting standard curve.

[0079] Table 15. Calculation table of relative error between calculated viable count and actual viable count of the unbroken direct measurement fitting standard curve (continued).

[0080] The results showed that the relative errors between the calculated and actual viable counts of *Bacillus subtilis* cultured in shake flasks and reactors were both less than 10% when using standard curves to fit protein concentration and viable count after lysis and centrifugation, and when using standard curves to fit protein concentration and viable count after direct measurement without lysis. This indicates that both standard curves are universally applicable to *Bacillus subtilis* cultured in different volumes. Furthermore, the relative error calculation results showed that the calculated and actual viable counts were even lower for all samples under both curves after 4-fold concentration treatment. This indicates that the fitted curves are more accurate for viable count detection when *Bacillus subtilis* cultured in different volumes undergo 4-fold concentration treatment. Therefore, protein concentration should be determined after 4-fold concentration treatment of the culture. The relative errors between the calculated and actual viable counts of the samples after 4-fold concentration treatment are summarized in Table 16.

[0081] Table 16 Results of viable cell count detection for *Bacillus subtilis* broth samples with different culture volumes using two standard curves.

[0082] In summary, by using two fitted standard curves to calculate the viable count of *Bacillus subtilis* cultured in shake flasks and reactors, the relative error between the calculated and actual viable counts was less than 10%, and the relative error between the calculated and actual viable counts was even smaller for direct measurement without rupture.

[0083] Example 10: Method Detection and Repeatability Validation To verify the repeatability of the direct testing method without breakage determined in Example 9, five samples of Mycobacterium bovis liquid were used. Each sample was concentrated four times according to the method described in Example 1, and the viable count and actual viable count were calculated in three repeated tests. The results are shown in Table 17.

[0084] Table 17. Repeatability verification results of calculated viable cell count and actual viable cell count.

[0085] The repeatability verification results of the above samples show that the CV values ​​of the calculated viable count for each of the 5 samples (3 replicates) are all <3%, the CV value of the actual viable count is <10%, and the relative error between the calculated viable count and the actual viable count is <10%, indicating that the method of the present invention has good repeatability and stability.

[0086] This invention provides a method for quantitatively detecting the number of viable Mycoplasma bovis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for quantitatively detecting the number of viable bovine mycoplasma bacteria, characterized in that, Includes the following steps: The bovine mycoplasma bacterial suspension was centrifuged and resuspended to obtain a resuspended bacterial suspension. The protein concentration was measured, and the bovine mycoplasma viable bacterial count was calculated based on the fitted standard curve of protein concentration and bovine mycoplasma viable bacterial count.

2. The method according to claim 1, characterized in that, The bovine mycoplasma bacterial solution is a bacterial solution stored at 2~8 ℃ for 0~24 h.

3. The method according to claim 1, characterized in that, The protein concentration of the resuspended bacterial solution was determined by either method (1) or (2) as follows: (1) Directly detect the protein concentration of the resuspended bacterial solution; (2) After the resuspended bacterial solution is broken, it is centrifuged again, and the supernatant is taken to detect the protein concentration.

4. The method according to claim 3, characterized in that, The aforementioned fragmentation is a non-contact, focused-energy ultrasonic fragmentation.

5. The method according to claim 4, characterized in that, The non-contact focused ultrasonic fragmentation method described herein has an ultrasonic fragmentation duration of 50-60 minutes.

6. The method according to claim 3, characterized in that, The second centrifugation is performed at 10000~12000 r / min for 10~20 min.

7. The method according to claim 3, characterized in that, When the protein concentration is obtained by direct detection of the resuspended bacterial solution, the fitting standard curve is shown in Equation I: y = 0.0106*x - 0.4478 (I), Where x is the protein concentration obtained by direct detection, in μg / mL; y is the viable cell count, in 10⁻¹⁰. 9 CFU / mL.

8. The method according to claim 3, characterized in that, When the protein concentration is obtained by centrifuging the resuspended bacterial culture again after disruption and then detecting the supernatant, the fitting standard curve is shown in Equation II: y=0.111*x1-0.2967 (II) Where x1 is the protein concentration obtained by centrifugation again after disruption, measured in μg / mL; y is the viable cell count, 10-1. 9 CFU / mL.

9. The method according to claim 1, characterized in that, The bovine mycoplasma bacterial solution includes a bacterial solution obtained through bovine mycoplasma fermentation culture.

10. The method according to claim 1, characterized in that, The resuspension mentioned refers to resuspension using PBS buffer.

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