9.3.6. Multiple assays

With careful primer design (see section 9.3.1.) it should be possible to approach 100% correct detection (no false positive or false negative results) for most viruses with a single primer pair. This is, however, very much conditional on the natural variation and variability (i.e. the capacity to generate new variants) for each virus. There are valid arguments that PCR is perhaps too specific for the reliable detection of highly variable entities such as RNA viruses, even when employing several different primer sets (Gardner et al., 2003). When the reliability of a primer set with respect to virus variability is in doubt, the best resolution is to employ several primer sets in parallel so that the failure of one set does not necessarily result in misdiagnosis. Multiple primer sets also allows one to estimate the rate of misdiagnosis by different primer sets due to virus variability (Chui et al., 2005). Within the honey bee viruses, multiple primer sets may be needed for reliable diagnosis within the highly variable ABPV complex (de Miranda et al., 2010a) and the slightly less variable DWV-VDV-1 complex (de Miranda and Genersch, 2010). Multiple assays are available for most honey bee viruses, and comparisons of multiple assays have been made for SBV (Grabensteiner et al., 2001), ABPV (Bakonyi et al., 2002a; 2002b) and DWV (Genersch, 2005).