9.3.4. Protocols

Numerous qualitative and quantitative RT-(q)PCR protocols have been published for the honey bee viruses (Annex 1), although few have been optimized experimentally. The European Reference Laboratory for bee diseases at ANSES (France) is in the process of designing fully optimized, validated RT-qPCR protocols for all bee viruses (see Blanchard et al., 2012) for routine, standardised diagnostic use by accredited laboratories. For experimental purposes, existing published protocols can be used and optimized, many of which can be easily adapted to qPCR using SYBR-green dye detection. Alternatively, new protocols can be designed based on the following practical, robust protocols for Reverse Transcription, One-Step RT-qPCR and Two-Step RT-qPCR, suitable for either quantitative or qualitative analyses. These provide a useful basis for individual adaptation and optimization, using the guidelines given above and in the BEEBOOK paper on molecular techniques (Evans et al., 2013).