8.2. DNA extraction
Cellulose-based affinity columns such as QIAGEN®, or generic equivalents are most practical for obtaining clean DNA preparations. They are reliable and yield good quality DNA. Magnetic bead-based purification also works well (e.g. Budge et al., 2010). Since samples of adult bees contain more secondary metabolites and phenolics than larvae, including a QiaShredder in the protocol will yield purer nucleic acid (DNeasy® Plant Mini Kit) and prevent inhibition of the PCR reaction. This is also recommended when extracting bacterial DNA from honey. The columns can be used for manual DNA extraction or in a QiaCube (QIAGEN®) for automated extraction. There are two options when considering extraction controls for the quantification of M. plutonius in honey bee samples. First, it is possible to monitor extraction efficiency using a honey bee reference gene (e.g. 18S; Budge et al., 2010). Alternatively extraction failures or PCR amplification inhibition can be monitored by amending the sample with a known amount of Staphylococcus aureus before extraction (Grangier, 2011). It is also recommended to include a negative extraction control (e.g. water) to check for possible contamination during the extraction process (Bustin et al., 2009). For further information on nucleic acid extraction see the molecular methods paper of the BEEBOOK (Evans et al., 2013).