10.2.3. Tagged cDNA primers

To further improve strand-specific detection of viral RNA, tagged RT-PCR can be used (Craggs et al., 2001). This method relies on a primer for cDNA synthesis which contains a tag sequence at the 5’-end that is unrelated to either virus or host. PCR amplification is then carried out with a primer consisting of only the tag sequence, together with a virus-specific upstream primer. This ensures that only cDNA’s derived from the tagged cDNA primer are amplified, and not cDNAs from false-, self- or mis-priming events. It is therefore important to ensure that the chosen tag sequence does not show any homology with a known bee pathogen or invertebrate sequence, by checking the tag sequences against the nucleotide sequence databases available on the NCBI website, using BLAST (Altschul et al., 1990). Tag sequences screened for use with honey bee viruses are shown in Table 5.