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

Abstract

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 30–3 × 10 6 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 10 4-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.