2.6. Comparison of detection limits between the RT-LAMP and the
real-time RT-PCR assays with RNA from cell cultures The detection limits of the RT-LAMP assays for detection of 3D region of FMDV genome were assessed using epithelial suspensions of O1Manisa (TUR/8/69) and SAT2/BOT/16/2008. Template RNA was extracted using a Magna Pure LC (Roche), as described above. Ten-fold serial dilutions of template RNAs were prepared using RNAse-free distilled water (Ambion, Grand Island, NY, USA). Multiplex RT-LAMP reactions were setup in duplicate as previously described using 2 l of template RNA per reaction. In addition, the performance of each of the component RT-LAMP assays was assessed under these conditions using nuclease-free water to substitute for the second set of primers in the reaction. The detection limits were defined as the highest dilutions at which both samples tested positive using the four multiplex RT-LAMP assays (as well limit of the real-time RT-PCR assay was also determined using 5 l of the same template RNAs (in duplicate) as described above