A bovine respiratory pathogen preservation solution, a preparation method and application thereof
By preparing a bovine respiratory pathogen preservation solution containing specific components, the integrity of bovine respiratory pathogens during preservation and transportation has been solved. This enables room temperature preservation and transportation, ensuring the integrity of the pathogens and the accuracy of detection, while reducing detection costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING BIAOCHI ZEHUI BIOTECHNOLOGY CO LTD
- Filing Date
- 2022-10-27
- Publication Date
- 2026-05-29
Smart Images
Figure BDA0003911405450000011 
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Figure BDA0003911405450000022
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological preservation reagent technology, specifically relating to a bovine respiratory pathogen preservation solution, its preparation method, and its application. Background Technology
[0002] Bovine respiratory disease (BRD) is a collective term for diseases such as bovine pneumonia, transport fever, and bronchitis caused by the interaction of multiple factors including the environment, the host itself, and pathogens. It is characterized by high morbidity, high mortality, and severe economic losses, and is a significant disease threatening the healthy development of the cattle industry worldwide. Pathogens causing bovine respiratory disease mainly include hemolytic Mansonia, Pasteurella multocida, Haemophilus influenzae, Cryptococcus pyogenes, Mycobacterium tuberculosis, and Mycoplasma. These pathogens often form mixed infections, thus requiring laboratory diagnosis to determine the pathogen type. Currently, swabs are typically used to collect pathogens from the surface of the bovine respiratory tract using a respiratory sampler. The swabs are then stored and transported at low temperatures in physiological saline or phosphate buffer, and subsequently isolated, cultured, and identified in the laboratory to determine the distribution and infection status of the bovine respiratory pathogens, allowing for targeted preventative and treatment measures.
[0003] However, during preservation and transportation, samples are typically required to be delivered to a testing laboratory within 4 hours at temperatures below 4°C. Most cattle farms are geographically remote, making it difficult to deliver samples within this timeframe, and the low-temperature transportation increases testing costs. Furthermore, prolonged preservation and transportation can lead to the death, inactivation, or nucleic acid degradation of collected pathogens (such as hemolytic Mansonia, Pasteurella multocida, Haemophilus influenzae, Cryptococcus pyogenes, Mycobacterium tuberculosis, and Mycoplasma). It can also cause contamination by other microorganisms, affecting subsequent isolation, culture, and identification, thus impacting the misjudgment or misdiagnosis of bovine respiratory pathogen distribution. Therefore, to accurately detect the distribution and infection status of bovine respiratory pathogens and implement targeted prevention and treatment measures, there is a need to develop a bovine respiratory pathogen preservation solution. However, there are currently no reports on bovine respiratory pathogen preservation solutions in existing technologies. Summary of the Invention
[0004] To address one or more problems existing in the prior art, one aspect of the present invention provides a bovine respiratory pathogen preservation solution comprising the following components at concentrations (final concentrations in the preservation solution):
[0005]
[0006]
[0007] In some embodiments, the pH value of the bovine respiratory pathogen preservation solution is 7.2-7.6.
[0008] Another aspect of the present invention provides a composition for preparing a bovine respiratory pathogen preservation solution, comprising the following components:
[0009]
[0010] In another aspect, the present invention provides a method for preparing a bovine respiratory pathogen preservation solution, comprising: dissolving sodium carboxymethyl cellulose, gentamicin, fetal bovine serum, tryptone, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in pure water according to the required concentration of each component, and making up to a final volume to obtain the bovine respiratory pathogen preservation solution.
[0011] In some embodiments, after dissolving the components in pure water, the step of adjusting the pH of the resulting solution to 7.2-7.6 is also included.
[0012] In some embodiments, after adjusting the volume, the solution is further filtered; preferably, a 0.22 μm filter membrane is used to filter the solution after adjusting the volume.
[0013] Another aspect of the present invention provides a method for the preservation and transportation of biological samples, which includes placing the collected biological samples in a tube containing the above-mentioned bovine respiratory pathogen preservation solution or a preservation solution prepared from the above-mentioned composition, sealing the tube, and then preserving and / or transporting the samples.
[0014] In some embodiments, the biological samples include bovine sputum, saliva, and bovine respiratory swab collection fluid.
[0015] The application of the bovine respiratory pathogen preservation solution or the composition described herein in the preparation of a kit for detecting bovine respiratory pathogens is also within the scope of this invention.
[0016] In another aspect, the present invention provides a method for identifying bovine respiratory pathogens, comprising the following steps:
[0017] 1) Biological samples collected from the bovine respiratory system are placed in tubes containing the above-mentioned bovine respiratory pathogen preservation solution or a preservation solution prepared from the above-mentioned composition, sealed, preserved, and transported to the testing laboratory; and
[0018] 2) Identify bovine respiratory pathogens in the tubes in the testing laboratory.
[0019] In some embodiments, the bovine respiratory pathogens include hemolytic Mansonia, Pasteurella multocida, Haemophilus stomatitidis, Cryptococcus pyogenes, Mycobacterium tuberculosis, or combinations thereof.
[0020] The bovine respiratory pathogen preservation solution provided by the above technical solution contains appropriate amounts of sodium carboxymethyl cellulose, gentamicin, fetal bovine serum, tryptone, phosphate, and sodium chloride. Sodium carboxymethyl cellulose has a chelating effect, uniformly adsorbing the collected pathogens and preventing interactions between them. Gentamicin prevents the growth of pathogens or other bacteria. Fetal bovine serum and tryptone protect the surface proteins of pathogens. Phosphate, as a buffer, prevents the pathogens from swelling under hypotonic conditions or shrinking and dying under hypertonic conditions. The combined effect of these components provides excellent protection against bovine respiratory pathogens, ensuring the integrity of the collected pathogens and preventing contamination by other bacteria. This facilitates subsequent pathogen isolation, culture, and identification, enabling accurate detection and identification of the distribution and infection status of bovine respiratory pathogens, and allowing for targeted preventative and treatment measures. Furthermore, this preservation solution allows for sample preservation and transportation at room temperature, saving costs. Detailed Implementation
[0021] The present invention aims to provide a preservation solution that can provide good preservation effect for bovine respiratory pathogens collected from the bovine respiratory tract, and provides a method for preparing the preservation solution and a method for using the preservation solution.
[0022] The present invention will be described in detail through the following specific embodiments.
[0023] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.
[0024] The embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be regarded as limiting the content of the present invention.
[0025] Example 1: Preparation of bovine respiratory pathogen preservation solution
[0026] Based on in-depth research into bovine respiratory pathogens, the inventors identified several important functional components in the preservation solution, including:
[0027] Chelating agents: used to adsorb collected pathogens and prevent interactions between pathogens;
[0028] Protein preservatives: used to prevent the destruction of the protein structure on the surface of pathogens, thereby preventing the degradation of pathogen nucleic acids;
[0029] Antimicrobial agents: used to prevent the growth and contamination of pathogenic bacteria or other microorganisms;
[0030] Stabilizers: Used to maintain pH stability and prevent pathogens from swelling in low osmotic pressure or shrinking and dying in high osmotic pressure.
[0031] Based on the above considerations, the inventors, taking into account the coordination and combination possibilities among the various functional components, prepared numerous preservation solutions and determined a suitable bovine respiratory pathogen preservation solution formula. Table 1 below shows some of the preservation solutions (formulas 1-6). The preparation process of the preservation solutions in Table 1 is as follows:
[0032] 1) Accurately weigh the required amounts of sodium carboxymethyl cellulose, fetal bovine serum, tryptone, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and casein peptone according to the ingredients in the formula described in Table 1 below, dissolve them in 800ml of pure water, add the required amount of gentamicin or vancomycin, stir at 220rpm for 30min, and mix thoroughly.
[0033] 2) Adjust the pH of the solution obtained in step 1) to 7.4 using NaOH (0.01M) or dilute HCl;
[0034] 3) Add the pH-adjusted solution from step 2) to a 1000ml volumetric flask and dilute to 1000ml with pure water;
[0035] 4) Filter the solution after adjusting the volume in step 3) through a 0.22μm filter membrane to obtain the preservation solution corresponding to each formula, and dispense it into 10ml centrifuge tubes for storage at room temperature.
[0036] Table 1: Preservative Solution Formulation
[0037]
[0038] Example 2: Test on the preservation effect of preservation solution on bovine respiratory pathogens
[0039] This embodiment examines the preservation effects of the preservation solutions prepared in Examples 1-6 on bovine respiratory pathogens. Cattle diagnosed by the laboratory as having bovine respiratory infection with Pasteurella multocida were selected as the test subjects. The specific procedures included the following:
[0040] (2.1) Collect bovine respiratory surface fluid using a sampling swab through a bovine respiratory sampler and place it into 10ml centrifuge tubes containing preservation solutions of formulations 1-6 prepared in Example 1. Cap the centrifuge tubes, shake gently, place the centrifuge tubes in a sample collection box, and transport them to the laboratory at room temperature (the sample preservation and transportation time takes about 12 hours). Each formulation preservation solution is performed in 3 replicates.
[0041] (2.2) Perform microscopic examination, analysis of contamination, pathogen isolation and nucleic acid detection according to conventional pathogen (e.g., bacterial pathogen) testing methods (among which, the PCR detection method for Pasteurella multocida established by Yu Xinyou et al. (Yu Xinyou et al., Establishment and application of dual PCR dual detection method for Pasteurella multocida and Mycoplasma bovis, Chinese Dairy Cattle, 2015(14)) to detect Pasteurella multocida in the respiratory tract of the cattle) to determine the status of respiratory infection with Pasteurella multocida in the cattle.
[0042] The results of pathogen testing for *Pasteurella multocida* in samples from preservation solutions of formulations 1-6 are shown in Table 2 below. *Pasteurella multocida* was detected and isolated from samples from formulations 1-3 (by microscopic examination and nucleic acid detection), and no other bacterial contamination was detected. *Pasteurella multocida* was neither isolated nor detected in samples from preservation solution 4; instead, other bacterial contamination was detected. This may be because preservation solution 4 cannot effectively prevent bacterial contamination, which could lead to the death and inactivation of *Pasteurella multocida* in the samples. Furthermore, preservation solution 4 cannot effectively protect the integrity of the surface protein structure of *Pasteurella multocida*, leading to nucleic acid degradation. Formulation 5... Although *Pasteurella multocida* was not isolated or detected under a microscope in the samples from the preservation solutions, *Pasteurella multocida* was detected by nucleic acid testing (with a weak signal), and contamination by other microorganisms was also detected. This is likely because the preservation solution in Formula 5 cannot effectively prevent contamination by other microorganisms, which may lead to the death and inactivation of *Pasteurella multocida* in the samples, although some *Pasteurella multocida* nucleic acid remained undegraded. *Pasteurella multocida* could be isolated and detected in the samples from the preservation solution in Formula 6, but the signal of the detected *Pasteurella multocida* was weak (possibly because the surface protein structure of some *Pasteurella multocida* was damaged, leading to nucleic acid degradation), and no contamination by other microorganisms was detected. Based on these results, the preservation solutions in Formulas 1-3 can be identified as the bovine respiratory pathogen preservation solutions of this invention. They can effectively prevent contamination by other microorganisms in biological samples collected from the bovine respiratory system and can ensure the integrity of the pathogens present in the biological samples, protecting the integrity of the pathogen nucleic acid, thus facilitating subsequent isolation and identification of the pathogens.
[0043] Table 2: Test results of Pasteurella multocida in samples from preservation solutions of formulations 1-6
[0044]
[0045] Example 3: Test on the preservation effect of preservation solution on various bovine respiratory pathogens
[0046] This embodiment examines the preservation effects of the preservation solutions prepared in Examples 1-3 on various bovine respiratory pathogens (Pasteurella multocida and Mycoplasma bovis). Cattle diagnosed by the laboratory as having co-infection with Pasteurella multocida and Mycoplasma bovis were selected as the test subjects. The specific procedures included the following:
[0047] (3.1) Collect bovine respiratory surface fluid using a sampling swab through a bovine respiratory sampler and place it into 10ml centrifuge tubes containing preservation solutions of formulations 1-3 prepared in Example 1. Cover the centrifuge tubes, shake gently, place the centrifuge tubes in a sample collection box, and transport them to the laboratory at room temperature (the sample preservation and transportation time is about 12 hours). Each formulation preservation solution is performed in 3 replicates.
[0048] (3.2) Microscopic examination, contamination analysis, bacterial isolation and nucleic acid detection were performed according to conventional pathogen testing methods (among which the double PCR detection method established by Yu Xinyou et al. (Yu Xinyou et al., Establishment and application of double PCR double detection method for bovine Pasteurella multocida and bovine Mycoplasma, Chinese Dairy Cattle, 2015(14)) was used to detect bovine Pasteurella multocida and bovine Mycoplasma) to determine the respiratory tract infection status of the cattle with bovine Pasteurella multocida and bovine Mycoplasma.
[0049] The results of pathogen testing for Pasteurella multocida and Mycoplasma bovis in the samples from the preservation solutions of Formulas 1-3 are shown in Table 3 below. Pasteurella multocida and Mycoplasma bovis were detected (by microscopic examination and nucleic acid detection) and isolated from the samples from the preservation solutions of Formulas 1-3, and no contamination by other bacteria was detected. This is consistent with the laboratory diagnostic results. The experiment in this example proves that the preservation solutions of Formulas 1-3 can effectively prevent contamination by other bacteria in the samples, and can ensure the integrity of multiple pathogens present in the samples and prevent the degradation of pathogen nucleic acids.
[0050] Table 3: Detection results of Pasteurella multocida and Mycoplasma bovis in samples from preservation solutions of formulations 1-3
[0051]
[0052]
[0053] Based on the results of the above embodiments, the present invention provides a bovine respiratory pathogen preservation solution that can effectively protect the integrity of bovine respiratory pathogens and prevent contamination by other bacteria. This solution may contain the following components at the following concentrations: 10.0-20.0 g / L sodium carboxymethyl cellulose, 0.5-1.5 U / L gentamicin, 3.0-7.0 g / L fetal bovine serum, 8.0-12.0 g / L tryptone, 4.0-5.0 g / L sodium dihydrogen phosphate, 6.0-7.5 g / L disodium hydrogen phosphate, and 3.5-5.5 g... The combination of sodium chloride at a concentration of / L and other appropriate amounts of specific components can effectively preserve bovine respiratory pathogens. Under normal temperature conditions, the integrity of the collected bovine respiratory pathogens can be guaranteed, and contamination by other microorganisms can be prevented. This facilitates subsequent pathogen isolation, culture, and identification, enabling accurate detection and identification of the distribution and infection status of bovine respiratory pathogens, and allowing for targeted prevention and treatment. Furthermore, using this preservation solution allows for the preservation and transportation of samples at room temperature, saving costs and extending the preservation time.
[0054] Example 4: Application of bovine respiratory pathogen preservation solution
[0055] In this embodiment, the preservation solutions prepared in Formulas 1-3 of Example 1 (in 10ml capped plastic tubes) were used to preserve 10 swab samples collected from bovine respiratory tracts and transported to the testing laboratory at room temperature. After preservation for 8 hours, 16 hours, and 24 hours, the samples were examined under a microscope, isolated and cultured for pathogens, and tested for nucleic acid according to conventional pathogen testing methods. (When performing nucleic acid testing, the PCR detection method for hemolytic Mansonia solani established by Xu Hui et al. (Xu Hui et al., Development and Application of PCR Detection Kit for Hemolytic Mansonia solani, Progress in Animal Medicine, 2009, 30(1)13-17) can be used to detect any possible hemolytic Mansonia solani.) The PCR detection method for Pasteurella multocida (Yu Xinyou et al., Establishment and application of dual PCR detection method for Pasteurella multocida and Mycoplasma bovis, Chinese Dairy Cattle, 2015(14)) was used to detect Pasteurella multocida in possible cases; Mycoplasma bovis real-time fluorescent PCR detection kit (purchased from Inner Mongolia Jinmaishi Biotechnology Co., Ltd.) was used to detect Mycoplasma bovis in possible cases. The respiratory tract infection pathogens of the cattle from these 10 swab samples were determined. The detection results of the samples after 8 hours, 16 hours and 24 hours of preservation are shown in Tables 4, 5 and 6 below (the detection results of the preservation solution of Formula 2 are representative).
[0056] Table 4: Test results of 10 swab samples after being stored at room temperature for 8 hours
[0057]
[0058]
[0059] Table 5: Test results of 10 swab samples after 16 hours of storage
[0060]
[0061] Table 6: Test results of 10 swab samples after 24 hours of storage
[0062]
[0063]
[0064] As shown in Tables 4-6 above, the test results of the 10 swab samples after being stored at room temperature for 8 hours, 16 hours and 24 hours in Formula 2 bovine respiratory pathogen preservation solution were consistent. This indicates that the bovine respiratory pathogen preservation solution has a good preservation effect on bovine respiratory pathogens. The pathogens can be stored at room temperature for a long time (e.g., more than 24 hours) in the preservation solution, which is beneficial for the preservation and transportation of collected samples and will not damage the integrity of the pathogens. Of the 10 swab samples tested, *Pasteurella multocida* was detected and isolated from sample 1 (by microscopic examination and nucleic acid testing), indicating that sample 1 originated from a bovine respiratory tract infection caused by *Pasteurella multocida*. *Mannella hemolyticus* was detected and isolated from sample 2, indicating that sample 2 originated from a bovine respiratory tract infection caused by *Mannella hemolyticus*. *Pasteurella multocida* and *Mycoplasma bovis* were detected and isolated from sample 3, indicating that sample 3 originated from a bovine respiratory tract infection caused by *Pasteurella multocida* and *Mycoplasma bovis*. *Pasteurella multocida*, *Mannella hemolyticus*, and *Mycoplasma bovis* were detected and isolated from sample 5, indicating that sample 5 originated from a bovine respiratory tract infection caused by *Mannella multocida*. Samples 8 and 9 were found to be infected with Pasteurella multocida, hemolytic Mansonia solani, and Mycoplasma bovis. The presence and isolation of Pasteurella multocida and hemolytic Mansonia solani in sample 8 indicates that the cattle from sample 8 were infected with these pathogens. Sample 10 was found to contain Mycoplasma bovis, indicating that the cattle from sample 10 were infected with these pathogens. Samples 4, 6, 7, and 9 were not found to contain these pathogens, indicating that the cattle from these samples were not infected with these pathogens. Therefore, based on these test results, targeted treatment can be administered to the cattle, and targeted preventative measures can be taken for the herd.
[0065] The test results for Formula 1 and Formula 3 are the same as those in Tables 4-6 and will not be repeated here. They also serve as mutual verification of the accuracy of the test results for Formula 2.
[0066] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A bovine respiratory pathogen preservation solution, comprising the following components at a final concentration:
2. The bovine respiratory pathogen preservation solution according to claim 1 has a pH value of 7.2-7.
6.
3. A composition for preparing a bovine respiratory pathogen preservation solution, comprising the following components in an amount sufficient to prepare 1 L of bovine respiratory pathogen preservation solution:
4. The method for preparing the bovine respiratory pathogen preservation solution according to claim 1 or 2, comprising: Dissolve the required amounts of sodium carboxymethyl cellulose, gentamicin, fetal bovine serum, tryptone, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride in pure water according to the concentration of each component, and make up to a final volume to obtain the bovine respiratory pathogen preservation solution.
5. The preparation method according to claim 4, further comprising, after dissolving each component in pure water, adjusting the pH of the obtained solution to 7.2-7.
6.
6. The preparation method according to claim 4 or 5, further comprising, after adjusting the volume, a step of filtering the adjusted solution.
7. The preparation method according to claim 6, wherein the solution after volume adjustment is filtered using a 0.22 μm filter membrane.
8. A method for preserving and transporting biological samples, comprising placing the collected biological samples in a tube containing the bovine respiratory pathogen preservation solution of claim 1 or 2 or a preservation solution prepared from the composition of claim 3, and sealing the tube for preservation and / or transport.
9. The method of claim 8, wherein the biological sample is selected from one or more of the following: bovine sputum, saliva, and bovine respiratory swab collection fluid.
10. The use of the bovine respiratory pathogen preservation solution of claim 1 or 2 or the composition of claim 3 in the preparation of a kit for detecting bovine respiratory pathogens.
11. The application according to claim 10, wherein the bovine respiratory pathogen is selected from one or more of the following: hemolytic Mansonia, Pasteurella multocida, Haemophilus stomatitidis, Cryptococcus pyogenes, and Mycobacterium tuberculosis.