Table 4.

Isothermal amplification specifications

Amplification method	Supplier	Reaction mix (25 μl, total volume)	Incubation temperature Primers (°C)
LAMP (Loop-mediated amplification)	Eiken Chemical (Tochigi, Japan)	20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH2)SO4, 8 mM MgSO4, 0.1 % Tween 20, 0.8 M betaine, 8 U Bst DNA polymerase, 1.4 mM dNTPs, and 4.0 μM SYTO® 9 Green intercalating dye, 8 U RNase inhibitor	60–65	6 primers: FIP 1.6 μM BIP 1.6 μM Loop-F 0.8 μM Loop-B 0.8 μM F3 0.2 μM B3 0.2 μM
RPA (recombinase polymerase amplification)	TwistDX (Cambridge, UK)	Rehydration buffer 30 μl Freeze-dried reaction mix Template + H2O to 47.5 μl total volume, 4.0 μM SYTO® 9 Green intercalating dye; Add 2.5 μl of 280 mM MgActo initiate reaction, 8 U RNase inhibitor	37–40	2 primers: primer A 10 μM primer B 10 μM
NASBA (nucleic acid sequence based amplification)	Biomérieux (Durham, NC)	5 μl enzyme mix (RNase H, T7-RNA polymerase, reverse transcriptase), 10 μl NASBA buffer + 5 μl sample, 4.0 μM SYTO® 9 Green intercalating dye, 8 U RNase inhibitor	55–60	2 primers primer F 0.2 μM primer R 0.2 μM

Positive Controls and their primers are included in these kits

Reverse transcriptase is added to reaction mix for RNA targets, e.g., 0.63 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA)