标题：响应面法优化LAMP反应冻干保护剂配方的研究

      作者：蔡文凯 1，祝珊珊 1，郝宗杰 2，苏业俣 1，林艺平 3    1.厦门医学院药学系，福建 厦门 361000；2.武汉兰丁云医学检验实验室有限公司检验科，湖北 武汉 430000；3.厦门集美区疾病预防控制中心检验科，福建 厦门 361000

      卷次： 2024年35卷13期

      【摘要】 目的 筛选出适用于甲流病毒环介导等温扩增技术(LAMP)的最佳冻干保护剂组合，建立起可在室温下长时间储存的甲流病毒核酸检测体系，为甲流现场快速检测和分级诊疗提供技术支持。方法 以LAMP核酸扩增活性为评价指标，在单因素试验的基础上利用三因素三水平的响应面法，探究冻干保护剂(海藻糖、牛血清白蛋白和甘露醇)对LAMP试剂的保护效果并优化冻干保护剂的组合。结果 采用Box-Behnken试验结果进行回归模型拟合，LAMP试剂的保护剂最佳配方组合为7.64 wt%海藻糖、0.43 wt%牛血清白蛋白和0.85 wt%甘露醇。经真空冷冻干燥且加入保护剂后的LAMP试剂将以脱水形式贮存，可在常温下长期保持活性，使用时加水复溶即可。为了检验保护剂的稳定性，真空冷冻干燥后的LAMP试剂分别置于25℃和37℃下贮存，第0天、4天、8天、12天、16天、20天取出分别进行LAMP扩增活性检测。添加最优保护剂组合的LAMP冻干试剂其核酸扩增活性远大于添加单一保护剂或未添加冻干保护剂组。结论 添加最优保护剂组合能够延长LAMP试剂的贮存时间和保持试剂的稳定性。
      【关键词】 响应面分析；环介导等温扩增技术；真空冷冻干燥；冻干保护剂
      【中图分类号】 R446 【文献标识码】 A 【文章编号】 1003—6350（2024）13—1889—06

Optimization of freeze-dried protectants formulations for LAMP reactions using response surface methodology.CAI Wen-kai 1, ZHU Shan-shan 1, HAO Zong-jie 2, SU Ye-yu 1, LIN Yi-ping 3.
1. School of Pharmacy and PharmaceuticalScience, Xiamen Medical College, Xiamen 361000, Fujian, CHINA; 2. Department of Clinical Laboratory, WuHan LandingMedical Laboratory Co., Ltd, Wuhan 430000, Hubei, CHINA; 3. Department of Clinical Laboratory, Xiamen Jimei DistrictCenter for Disease Control and Prevention, Xiamen 361000, Fujian, CHINA
【Abstract】 Objective To screen the optimal combination of freeze-dried protectants suitable for the loop-medi-ated isothermal amplification (LAMP) reaction system, and establish a nucleic acid detection system for influenza A vi-rus that can be stored for a long time at room temperature, in order to provide technical support for rapid on-site detec-tion and graded diagnosis and treatment of influenza A virus. Methods LAMP nucleic acid amplification activity wasused as the evaluation index, the protective effect of freeze-dried protectants (alginate, bovine serum albumin, and manni-tol) on LAMP reagents was investigated and the combination of freeze-dried protectants was optimized using a three-fac-tor, three-level response surface methodology based on one-way test. Results A regression model was fitted to the re-sults of the Box-Behnken test, and the optimal combination of the protectants for LAMP reagent was 7.64 wt% alginate,0.43 wt% bovine serum albumin, and 0.85 wt% mannitol. The vacuum freeze-dried LAMP reagent with protective agentwill be stored in dehydrated form to maintain the activity for a long period of time at room temperature, and can bere-solubilised with water when used. In order to test the stability of protective agents, the vacuum freeze-dried LAMP re-agents were stored at 25℃ and 37℃, and then tested on days 0, 4, 8, 12, 16, and 20 for the detection of LAMP amplifica-tion activity. The nucleic acid amplification activity of the LAMP reagents with the optimal combination was much high-er than that of the single protectants or no protectants. Conclusion The optimal combination of freeze-dried protectantscan prolong the storage time and maintain the stability of the LAMP reagents.
      【Key words】 Response surface analysis; Loop-mediated isothermal amplification; Vacuum freeze-drying;Lyophilised protective agent