关键词: 牛支原体, 等温扩增, 冻干, 试剂盒, RT-PCR

Abstract: 【Objective】Mycoplasma bovis (M. bovis) is a pathogen related to a variety of syndromes of infected cattle, and it spreads widely in the world. For the survey of the epidemiology of M. bovis in China, simple and sensitive kits are needed.【Method】A recombinant plasmid with the uvrC gene of M. bovis was constructed and transfected into TOP10 competent cell, by which a strain of recombinant Escherichia coli was selected. The recombinant plasmid rP-uvrC was extracted, purified and diluted to the concentration of 10⁴ copies/μL, which was used as the positive control. Betaine solution and color developing solution (mixture of SYBR Green I and HNB) were prepared to solve the freeze-dried amplification reagent and visualize the result of the amplification reaction, respectively, according to the published reports. Three kinds of freeze-drying protective agent which did not inhibit the amplification reaction were selected from 8 agents generally used to produce vaccine, then they were combined by different ratios to form 27 combinations, and the best combination was selected according to the physical characters. The isothermal amplification reagent made with the best freeze-drying protective agents were transported to the lyophilizer to detect the eutectic point, and the best heating-up time of the first drying, the first and the second drying times were determined. The best lyophilizing curve was selected to lyophilized the isothermal amplification reagent by testing of the physical characters, vacuum and residual water content. The lyophilized isothermal amplification kit consists of one tube of lyophilized isothermal amplification agent, betaine solution, positive control and color developing solution. The sensitivity of the kit was determined with a series of rP-uvrC solutions (10⁰-10⁵ copies), and the specificity was determined with 10⁴ CCU·mL^-1 solutions of M.bovis strains PG-45, HB-1, SD-2, 10⁸ CCU·mL^-1solutions of M. bovirhinis and M. agalactiae, and 10⁸CFU·mL^-1 Pasteurella multocida and Tuberculosis mycobacteria, respectively. The steady of the kit was also evaluated under different temperatures.【Result】Among the 8 freeze-drying protective agents, only trehalose, mannitol and bovine serum albumin did not interfere the isothermal amplification reaction, and the best freeze-drying protective agents composition is 5% trehalose +1.25% mannitol +1.25% bovine serum albumin. The eutectic point was -16℃, and the best heating-up time of the first drying was 3h, the first and the second drying times are 6h and 4h, respectively. The detection limit of the freeze-dried kit is 10 copies, 10⁴ CCU·mL^-1 solutions of M.bovis strains PG45, HB-1 and SD-2 showed positive results, 10⁸ CCU·mL^-1 solutions of M. bovirhinis and M. agalactiae, 10⁸CFU·mL^-1 Pasteurella multocida and Tuberculosis mycobacteria showed negative results. The sensitivity of the kit didn't change even it was stored for 6 months under 20℃ or 10 d under 37℃. So, the shelf life of the freeze-dried kit could be about 24 months.【Conclusion】The above results suggest that the freeze-dried kit has higher sensitivity, specificity and stability, could be used as a point-of-care (POC) test product since it is convenient for storage and very easy for the veterinarians to use in the farms.

Key words: Mycoplasma bovis, loop-mediated isothermal amplification, freeze-drying, kit, RT-PCR