Detection of wheat dwarf virus (WDV) in wheat and vector leafhopper (Psammotettix alienus Dahlb.) by real-time PCR.

摘要

Wheat dwarf virus (WDV) is a newly emerging pathogen affecting wheat production in China. A real-time PCR method using the TaqMan probe is described for quantitative detection of WDV in wheat tissues and in leafhopper (Psammotettix alienus Dahlb.). Primers and probes for specific detection of WDV were designed within the conserved region of the coat protein (CP) gene sequence. A sensitivity assay showed the detection limit of the assay was 30 copies, and the standard curve was linear over range 30-3x106 copies, with good reproducibility. Simultaneously, this real-time PCR assay could be used to detect WDV CP genes in viruliferous leafhoppers. As determined by an end-point dilution comparison, real-time PCR was close to 104-fold more sensitive than the indirect enzyme-linked immunosorbent assay for WDV detection. Field samples of wheat and leafhopper collected from different regions of China were detected by both real-time PCR and gel-based PCR. The results showed more positive samples could be identified by real-time PCR than by gel-based PCR. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of WDV biology.Digital Object Identifier http://dx.doi.org/10.1016/j.jviromet.2010.07.029