Quantification of Honey Bee Viruses via Real-Time PCR

To quantify the virus abundance in the samples, all RNA extracts were analysed through Real-Time PCR using Power SYBR™ Green Cells-to-CT™ Kit (Thermo Fisher Scientific), as previously reported (Cilia et al., 2021 (link)). The primers used to amplify the target honey bee viruses considered here are reported in Table 3. The Real-Time PCR assay was performed on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific), following the protocols for each gene sequence. RNA previously extracted from positive honey bees was used as the positive control for each investigated virus. All the analyses were conducted in duplicates.
For each target, a standard curve was generated by amplifying the serially diluted recombinant plasmids containing the pathogen-specific RNA fragment from 1 * 10¹ to 1 * 10⁹ copies in a qPCR assay on a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific), as previously reported (Mazzei et al., 2019 (link); Cilia et al., 2021 (link)), following the amplification and quantification protocols (Chantawannakul et al., 2006 (link); de Miranda et al., 2010 (link); Kajobe et al., 2010 (link); Martin et al., 2012 (link); Hartmann et al., 2015 (link); Garigliany et al., 2017 (link); Mazzei et al., 2018 ).