8.3. PCR

When PCR is used solely for detecting the presence or absence of a specific DNA signature, it is referred to as qualitative PCR (yes or no answer). The qualitative PCR detects only the end product whereas the real-time PCR detects the amplicon as it accumulates and determines the number of new DNA molecules formed in each reaction. The amount of the target molecule can be quantified (qPCR) either relatively or as absolute values or numbers (for further general information see the molecular methods paper of the BEEBOOK (Evans et al., 2013)).

Four protocols for the detection and quantification of M. plutonius using PCR have been published to date (Table 2). Two protocols for qualitative PCR; one for detection in diseased larvae (Govan et al., 1998) and a hemi-nested PCR assay (Djordjevic et al., 1998).The latter method was further developed for the detection of M. plutonius in larvae, adult bees, honey and pollen (McKee et al., 2003; see section 8.3.1). The results obtained indicate: 1. that the PCR assay is far more sensitive than culture; 2. that not all the M. plutonius detected is viable or amenable to culturing; and 3. that honey samples may be a useful tool for detecting sources of M. plutonius.

Real-time PCR assays for the quantification (qPCR) of M. plutonius (Roetschi et al., 2008; Budge et al., 2010) have been used to analyse pooled samples of brood nest workers from several colonies within an apiary as a suggested alternative to routine visual brood control (Roetschi et al., 2008). However, more recent results suggest the amount of M. plutonius in adult bees provides a less stable estimate of the likelihood of finding disease than using larvae (Budge et al., 2011). The qPCR method can also be used to attribute a risk of EFB infection to collected samples measured as probability of the sample showing clinical symptoms and providing a trigger for later inspection of apiaries at risk (Budge et al., 2010; Grangier, 2010). This may provide a definitive diagnosis of EFB, based on a combination of the presence of clinical disease and the confirmed presence of M. plutonius. However, in some territories, the costs of such preliminary screening using real-time PCR may not be economically viable (Grangier, 2011).

Table 2. PCR-based methods for the detection of M. plutonius.

Table 2