2.4 Optimization and evaluation of the LAMP assays
The LAMP reaction was conducted in a LAMP reaction mixture containing 12 μl of 2× LAMP buffer [40 mM Tris-HCl, pH 8.8, 20 mM KCl, 16 mM MgSO4, 20 mM (NH4)2SO4, 0.2% Tween 20, 1.6 M Betaine and 2.8 mM of each dNTP], The outer primers and inner primers were optimized from 50 nM to 600 nM. 1.0 μl Bst DNA polymerase (New England Biolabs, MA, USA), 2.0 μl of extracted DNA and 4 μl of distilled water. The reaction mixture was performed in ABI QuantStudio 5. In addition, the LAMP reaction temperature was assessed by testing temperatures between 61oC and 67oC.