海藻糖对冷冻干燥的LAMP反应体系的保护效果

The protective effects of trehalose on freeze-drying and LAMP reagents storage

中文关键词: 真空冷冻干燥  环介导等温扩增  LAMP  试剂保存  海藻糖

英文关键词: Vacuum freeze-drying  LAMP  Trehalose  Reagents storage

中文摘要:

      环介导等温扩增（LAMP）技术近年来被广泛应用于病原生物的检测。为了解决LAMP反应试剂混合物在室温条件下的储运问题，本研究对添加5%、10%和15%海藻糖（质量/体积，g/ml）的LAMP反应试剂混合物进行冷冻干燥处理，研究了其在4℃、25℃和37℃储藏条件下活性维持的情况。研究结果表明，添加15%海藻糖能最大程度地保护LAMP反应试剂混合物的活性。相对于未经处理的普通LAMP体系混合物而言，添加15%海藻糖并经冷冻干燥的试剂混合物在4℃、25℃和37℃储藏条件下的活性半衰期分别为112.3、36.2和6.2 d，分别为对照组的6.5、8.7和2.1倍。在LAMP反应试剂混合物中添加海藻糖并进行冷冻干燥处理成本不高、方法简单，将有助于推动LAMP方法在各领域的大规模推广应用。

英文摘要:

      With the unique advantages of high sensitivity, equipment independence, and rapid procedure, loop-mediated isothermal amplification (LAMP) has been extensively developed to detecting various pathogens. The substances of enzyme, dNTP, and other complex organic molecules in LAMP mixture are subjected to a variety of chemical changes under room temperature, many of which are quite unacceptable in terms of storage and transportation. In order to solve this problem, we tried to develop a lyophilization technology for the protection of the LAMP mixture with the addition of trehalose as a stabilizer. The predictive effects of freeze-drying and different concentrations of trehalose (5%, 10% and 15%) in LAMP mixture during storage under temperature of 4℃, 25℃ and 37℃ on amplification activity of LAMP were analyzed systematically. The non-freeze-dried reagents without trehalose were also stored at the same temperature as controls. Our results showed that the lyophilizing LAMP reagents with 15% trehalose exhibited the greatest performance. Furthermore, at 4℃, the half-life of freeze-dried mixtures with 15% trehalose (w/v) was 112.3d, which was 6.5-fold of the control. At the temperature of 25℃, the half-life of treated reagents was 36.2d, which was 8.7-fold higher than control. Even under the temperature of 37℃, the half-life was 6.2d, which was 2-fold of the control. We report here the cost effective, simple developing procedure of a freeze-drying LAMP reagent which will promote the widespread use of LAMP in the field.