响应面法优化LAMP反应冻干保护剂配方的研究

Optimization of freeze-dried protectants formulations for LAMP reactions using response surface methodology

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。

Objective To screen the optimal combination of freeze-dried protectants suitable for the loop-mediated isothermal amplification(LAMP)reaction system,and establish a nucleic acid detection system for influenza A virus that can be stored for a long time at room temperature,in order to provide technical support for rapid on-site detection and graded diagnosis and treatment of influenza A virus.Methods LAMP nucleic acid amplification activity was used as the evaluation index,the protective effect of freeze-dried protectants(alginate,bovine serum albumin,and mannitol)on LAMP reagents was investigated and the combination of freeze-dried protectants was optimized using a three-factor,three-level response surface methodology based on one-way test.Results A regression model was fitted to the results 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 agent will be stored in dehydrated form to maintain the activity for a long period of time at room temperature,and can be re-solubilised with water when used.In order to test the stability of protective agents,the vacuum freeze-dried LAMP reagents were stored at 25℃and 37℃,and then tested on days 0,4,8,12,16,and 20 for the detection of LAMP amplification activity.The nucleic acid amplification activity of the LAMP reagents with the optimal combination was much higher than that of the single protectants or no protectants.Conclusion The optimal combination of freeze-dried protectants can prolong the storage time and maintain the stability of the LAMP reagents.