The OP samples from the 2017 FMD outbreak were analysed using a multiplex one-step RT-PCR assay. The assay was performed using the TaqMan® Fast Virus 1-Step Kit (Life Technologies) and kit manual. We added 2 μL of eluate extracted in the manual extraction section to a Master Mix containing nuclease-free water, 4X TaqMan buffer, primers and TaqMan probe. The thermal cycler (BioRad, Hercules, California) was programmed as follows; reverse transcription at 50 °C for 5 min; polymerase activation and DNA denaturation at 95 °C for 20 s; two-step amplification for 40 cycles with denaturation at 95 °C for 3 s; annealing and plate read at 60 °C for 30 s.
We analysed the PCR reaction to observe any positive FMD identification from the OPF samples collected and to compare the results obtained to those from the positive high and low standard controls. Reactions were considered positive if the amplification was detected before 32.0 cycles [59 (link)].
Before sequencing, we removed the primers and deoxynucleoside by enzymatically adding the ExoSAP-IT PCR Product Cleanup Reagent (0.5 μl of Exo and 2 μl of SAP) to 10 μl of amplicons. The mix was incubated at 37 °C for 15 min and later 85 °C for 15 to activate Exo and SAP respectively, lastly, the product was held at 4 °C.
We analysed the PCR reaction to observe any positive FMD identification from the OPF samples collected and to compare the results obtained to those from the positive high and low standard controls. Reactions were considered positive if the amplification was detected before 32.0 cycles [59 (link)].
Before sequencing, we removed the primers and deoxynucleoside by enzymatically adding the ExoSAP-IT PCR Product Cleanup Reagent (0.5 μl of Exo and 2 μl of SAP) to 10 μl of amplicons. The mix was incubated at 37 °C for 15 min and later 85 °C for 15 to activate Exo and SAP respectively, lastly, the product was held at 4 °C.