Protocol cDNA synthesis for PRRSV ORF 2-7 PCR: Protocol RT-reaction For preparation of the RT mix the next components were combined in a pre-mix: Component pre-mix n = 1 n = 13 (PRRSV) RNA 7 µL Primer stock 10 µM Poly dT 2 µL 26 µL 10 mM dNTP mix 1 µL 13 µL For each sample 2 µL poly dT primer and 1 µL dNTP mix were mixed with 7 µL RNA. Samples were incubated for 5 minutes at 65°C (denaturation) and subsequently cooled on melting ice for at least 1 minute. A cDNA synthese mix was prepared : Component n = 1 n= 13 10X RT buffer 2 µL 26 µL 25 mM MgCL2 4 µL 52 µL 0.1 M DTT 2 µL 26 µL RNaseOUT (40 U/µL) 1 µL 13 µL SuperScript III RT (200 U/µL) 1 µL 13 µL 10µL cDNA syntheses mix was added to 10 µL RNA/pre-mix . After mixing, the tubes were centrifuged for a few seconds. Samples were incubated for 75 minutes at 50°C (cDNA syntheses) and subsequently for 5 minutes at 85°C (termination). After that, the samples were cooled on melting ice. Samples were centrifuged for a few seconds and to each cDNA sample 1 µL RNAse H was added and incubated for 20 minutes at 37°C. The samples were used directly in the PRRSV ORF 2-7 PCR or stored at -70°C.