2.2.2.3.3. Realtime PCR for quantification of N. apis and N. ceranae

Molecular quantification of N. apis and N. ceranae allows both relative quantification of the two Nosema species or their absolute quantification per bee or per sample, which can be of considerable interest for studies of the interactions between these two pathogens. However, molecular quantification requires use of a real-time PCR machine; these machines are currently relatively expensive and they require calibration with dilution series of N. apis and N. ceranae to generate accurate quantification. Several realtime PCR machines are on the market, each employing different fluorophore chemistries for molecular quantification. Here we present a method based on Forsgen and Fries (2010) that uses a BioRad MiniOpticon real-time PCR machine and EvaGreen chemistry for quantification as it has functioned well in several laboratories.