The Stability Protection Of Biological Molecules In Loop-mediated Isothermal Amplification Reagents

With the unique advantages of high sensitivity, equipment independenceand rapid procedure, Loop-mediated isothermal amplification (LAMP) hasbeen extensively developed to detecting various pathogens. Thesubstances of enzyme, dNTP, and other complex organic molecules inLAMP mixture are subjected to a variety of chemical changes under roomtemperature, even more, many of which are quite unacceptable in thecourse of storage and transportation. For solving the problem, wesystematically analyzed the protective effect during long-term storage oflyophilization, paraffin segregation and diatomite immobilization in thepresence of different amount of trehalose (5%,10%and15%) as astabilizer at4℃,25℃,37℃and45℃.1. The effect of freeze-drying on the thermal stability of LAMP reagentsAfter addition of5%,10%and15%trehalose(w/v) respectively tothe LAMP reagents (containing Bst DNA polymerase (large fragement),buffer, dNTP, primers, betain et al.),2h of lyophilization was imposed,then the yields of LAMP premix were monitored regularly when stored at 4℃,25℃and37℃for3month. Our results showed that the lyophilizedLAMP reagents with15%trehalose showed the greatest visibleperformance. Furthermore, compared with the controls (non-treatedreagents stored at the same temperature) the half-life of freeze-driedmixtures with15%trehalose was112.3d and36.2d at the temperature of4℃and25℃, which were5.9and7.7times more than that of the control.Even under the temperature of37℃, the half-life was increased by2-fold,which was6.2d. Lyophilization and the addition of trehalose to themixture extended the half-life of LAMP reaction mixture.2. The effect of paraffin segregation on the thermal stability of LAMPreagentsWe used paraffin film as a stable barrier to seal Bst DNA polymerase(large fragement) enzyme and Bst DNA polymerase storage buffer (8U BstDNA polymerase (large fragement),10mM Tris-HCl,1mMDithiothreitol,0.1mM EDTA,50%Glycerol,0.1%Triton X-100, pH7.5)with different amount of trehalose (0%or15%) under the PCR tubesbefore the addition of the other substance of LAMP reagents above. Thethermal stability of paraffin-sealed preparations were analysed in the termsof90d of storage at4℃,25℃and37℃. The results showed that, thehighest half-life time vaules were obtained in the presence of15%trehalose, as the yields of amplification of LAMP reagents with which were increased by30%compared with the control after stored at4℃and25℃. Further more, the half-life of segregated premix was increased by6.1-,16.2-and5.3-fold by adding15%trehalose respectively after kept atthe4℃,25℃and37℃temperature stresses. These results lead to theindication that paraffin segregation prolonged the half-life of labileenzymes by against mixing of aqueous reagents.3. The effect of diatomite immobilization on the thermal stability ofLAMP reagentsAfter the co-heating of Bst DNA polymerase (large fragement) storagebuffer and diatomite at55℃for30min, the other substances of LAMPreagents were added to the PCR tubes. The thermal stability ofimmobilized preparations were analysed in the terms of90d of storage at4℃,25℃,37℃and45℃. Our results showed that the best finalconcentration of trehalose in premix was15%. When15%trehalose wasadded, the half-lives of immobilited reaction mixtures were increased to17.0-,52-, even8.3-fold at the tested temperature of4℃,25℃and37℃respectively. Similar pattern of increase in thermal stability was observedwhile the immobilized preparation was under the extremely45°Ctemperature stress of45°C. These results suggested that diatomite had asignificant potential as a good stabilizer at higher temperatures.According to the results above from on-time monitoring, diatomite immobilization displays the best protective effect among the threedifferent means described above at all the tested temperature, both thethermal stability and the shelf stability of LAMP reagents are prolonged.The cost effective, simple developing procedure of three differenttreated LAMP reagents that we have developed here will promote thethermal stability of LAMP reaction mixtures during storage and transportunder high temperature stress.