Honey Bee Virus Quantification Protocol

Bees of each sample were placed in a 15 mL plastic bottle together with 5–10 steel bearing balls. Using a technique adapted from plant virology, the samples were freeze dried, homogenized in a genogrinder and thereafter, RNA was extracted according to the manufacturer’s manual (for details see [13 (link)]). Following RNA extraction, a two-step real-time RT-PCR assay was used to detect and quantify seven honey bee viruses, BQCV, CBPV, SBV, DWV, ABPV, KBV, and IAPV. The three closely related viruses of the ABPV complex (ABPV, KBV and IAPV) were detected in a single assay (AKI) [51 (link)]. The housekeeping gene, β-Actin, was used as an internal control, where the presence and quantification of this reference gene ensured that the entire procedure from extraction to quantification was done without degradation of RNA [13 (link)].
Quantitative PCR amplifications were carried out on a vii7 apparatus (Applied Biosystems) in duplicate for each sample using SYBR Green DNA binding dye. Final volumes of 12μL with a primer concentration of 0.4μM were loaded on optical 384 well PCR plates. Primers [30 , 47 (link), 51 (link)–53 (link)] used in this study are listed in Table 1.