Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

Li, Ji Lian; Cornman, R. Scott; Evans, Jay D.; Pettis, Jeffery S.; Zhao, Yan; Murphy, Charles; Peng, Wen Jun; Wu, Jie; Hamilton, Michele; Boncristiani, Humberto F.; Zhou, Liang; Hammond, John; Chen, Yan Ping

The amplification for both TRSV and ␤-actin was carried out following the manufacturer's recommended protocol for thermal profile parameters for three-step PCR. After amplification, a melting curve analysis was performed to determine the specificity of the PCR products. Each sample was run in triplicate, and the qPCR assay was repeated twice. The amplification efficiencies of the SYBR green real-time RT-qPCR assay for both TRSV and ␤-actin were proved to be approximately equal (data not shown). The output of RT-qPCR assays for TRSV in different tissues was interpreted by using the comparative cycle threshold method (⌬⌬C T method). The average C T value (⌬C T ) of TRSV in each tissue was normalized using the C T value corresponding to the endogenous control, ␤-actin, with the following formula: ⌬C T ϭ average C T (TRSV) Ϫ average C T (␤actin). The tissue that had the lowest level of TRSV was chosen as a calibrator. The ⌬C T value of each tissue was subtracted from the ⌬C T value of the calibrator to yield ⌬⌬C T . The concentration of TRSV in each tissue was calculated using the formula 2 Ϫ⌬⌬CT and expressed as n-fold difference relative to the calibrator.