Development of a real-time RT-PCR for the universal detection of LChV-1 and study of the seasonal fluctuation of the viral titer in sweet cherry cultivars

Little cherry virus 1 (LChV1) is a sweet cherry pathogen which has lately been reported in other Prunus species. LChV1 variability makes reliable detection a challenging undertaking. The objective of this work was to develop a rapid, sensitive and reliable one-tube, real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for the detection and quantification of LChV1. Primers and a TaqMan® probe were designed, using conserved regions of the capsid protein (CP) gene. Detection range was evaluated using several divergent viral isolates. The amplification efficiency of the method was estimated at 96.7%, whereas the detection limit was about 100 RNA copies. The protocol was applied in the study of virus fluctuation within leaves and phloem tissue throughout the year and better periods to test and plant tissues to sample were determined. Comparative analysis of this method with a previously published nested RT-PCR, revealed the higher analytical and diagnostic sensitivity of the new test ma...