*4.4. PCR Assays to Detect Virus RNA*

All RNA extracts were retro-transcribed by M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using a blend of oligo-d (T) primers and random hexamers following the manufacturer's instruction. Five microliters of the obtained cDNAs were used as a template for the PCR reactions, performed using HotStarTaqPlus Polymerase Mix (Qiagen). Primers to amplify the viral genomes of the honey bee viruses investigated herein are reported in Table 3. The real-time PCR assay was performed on a Rotorgene Corbett 6000. RNA extracted previously from positive honey bees was used as the positive control for each investigated virus.

**Table 3.** List of primers used to detect viruses in Aethina *tumida*.



**Table 3.** *Cont.*

KBV: Kashmir bee virus; DWV: Deformed wing virus; ABPV: Acute bee paraylis virus; IAPV: Israeli acute bee paryalis virus; BQCV: Black queen cell virus; SBV: Sac brood virus; CBPV: Chronic bee parylis virus; SPV: slow paralysis virus.
