Detecting Active Viral Infections in Bumblebees

Although bees may pick up viruses on flowers and test positive for the presence of a virus, these are not necessarily active infections. To test for actively replicating viruses in the bumblebees, we conducted strand specific RT-PCR [38 (link)] on extracted RNA samples that tested positive for a virus. Each RNA sample was transcribed to cDNA using iScript cDNA Synthesis Kit (BioRad). To increase specificity, we used PAGE purified, biotinylated forward and reverse primers (Integrated DNA Technologies) during reverse transcription and purified the resulting cDNAs using magnetic beads coated with a monolayer of streptavidin following manufacturers protocols (New England BioLabs). We diluted each cDNA tenfold and then conducted PCRs with non-biotinylated primers in separate reactions for both for forward and reverse strands.