Development of RealTime assay for the detection of Strawberry Crinkle Virus.

Viruses represent one of the major threats for the strawberry industry, causing important economic losses, and their control is based mainly on rigorous certification programs to maintain clean planting stocks; these viruses include SCV (Strawberry Crinkle Virus) the causal agent of a quarantine disease of major economic importance. All strawberry cultivars are susceptible to SCV infection but due to the absence of obvious symptoms, the detection is limited to indexing on strawberry indicator plants, an expensive and time-consuming procedure. The aim of this study is to develop a sensitive, reliable and fast RealTime assay, based on TaqMan chemistry and amplifying a portion of the conserved L gene sequence coding for RNA-dependent RNA polymerase (RdRp). As strawberry is a difficult host for virus detection, an evaluation of the efficiency of 4 different RNA extraction methods and a comparison between conventional nested-PCR and RealTime assay were carried out. RealTime detection of SCV in several symptomatic and asymptomatic strawberry plants, field collected, indexed on strawberry indicator plants and/or maintained under CAV's screen-houses, proved highly efficient and reproducible, and its sensitivity was as high as the nested PCR assay. This new protocol represents an improvement on the existing analytical methods and could be used as a reliable diagnostic procedure for routine tests in certification and control programs.