Magna pure lc total nucleid acid isolation kit

All OPF, heparinised blood, urine and faeces suspension samples were tested for presence of FMDV by plaque count on monolayers of secondary lamb kidney cells (virus isolation, VI). Samples were tested in 2 wells of a six-well plate using 200 μL per well, as previously described [20 (link)]. All OPF samples were also tested for presence of FMDV by RT-PCR. RNA isolation was performed using the Magna Pure LC total Nucleid Acid Isolation kit® (Roche) and the MagNa Pure 96 system® (Roche). Isolated RNA was tested in a LightCycler 480 Real-Time PCR System® (Roche) using a QuantiFast Probe RT-PCR kit® (Qiagen), all in accordance with the manufacturers’ instructions. The primers, probes and test protocol used have been previously described [21 (link)].

All oral swab, heparinised blood, urine, and faeces samples were tested for the presence of FMDV as described previously [11 (link)], using plaque titration on monolayers of secondary lamb kidney cells (VI, i.e. detection of infectious virus particles). In addition all oral swab and probang samples were tested for the presence of FMDV using real time RT-PCR because in these samples neutralising antibodies, that could influence the virus isolation results, were expected to be present. RNA isolation was performed using the Magna Pure LC total Nucleid Acid Isolation kit (03 038 505) in the MagNa Pure 96 system (Roche®, Mannheim, Germany). Isolated RNA was tested as described previously [19 (link)] using a LightCycler 480 Real-Time PCR System (Roche®) with the exception that we used a Quantifast Probe RT-PCR Kit (Qiagen®, Venlo, The Netherlands).