12.3.1. Method detection limit
The method detection limit (DLmethod) is the lowest amount of biological target in a sample that can be detected by the entire method (from processing through RT-qPCR). The DLmethod is evaluated with biological reference samples obtained by spiking virus-free bee homogenates with known amounts of purified virus. At least two independent trials must be performed on two independent two-fold serial dilutions, with four replicate RNA extractions at each dilution level. The DLmethod is the last dilution at which viral RNA can be detected in all replicates (100% frequency).