Figure 3

RT-PCR incorporated with biotinylated primer and purification of magnetic beads for strand-specific detection of DWV RNA. Total RNAs extracted from bees with deformed wings. The negative and positive-strand cDNAs that were generated by biotinylated forward (Lane 2 and 6) and reverse primers (Lane 3 and 7) in reverse transcription, respectively. The cDNA was generated by one step RT-PCR with both biotinylated forward and reverse primers (Lane 4 and 8). Reverse transcription was conducted without the addition of primer (Lane 5 and 9). The biotinylated cDNAs were either amplified directly by PCR (Lane 2-5) or subjected to magnetic bead purification before PCR amplification (Lane 6-9). Negative control without template (Lane 10) and positive control with recombinant DWV plasmid DNA (Lane 11) were included in the reaction. A 100-bp ladder was loaded into lane 1. The arrow on the right indicates the expected 702 bp RT-PCR products.