A one-step real time RT-PCR assay for quantifying rice stripe virus in rice and in the small brown planthopper...

Rice stripe virus (RSV) is an important pathogen affecting rice production in subtropical and temperate regions. One-step real time RT-PCR methods using the TaqMan probe are described for quantitative detection of RSV in rice tissues and in Laodelphax striatellus Fallen, the small brown planthopper (SBPH). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein (CP) gene sequence. A DNA fragment was amplified for mimicking the complementary RNA by PCR-based gene assembly, and was used for generation of standard RNA templates. A sensitivity assay showed that the detection limit of the assay was 20 copies, and the standard curve had a linear range from 20 to 2 x 10(5) copies, with good reproducibility. An internal control assay designed to target the rice ubiquitin 5 gene (UBQ5) was included in detecting RSV in different infected rice tissues. Simultaneously, a real time RT-PCR assay was used to detect the RSV CP gene in viruliferous SBPH. The results showed that the numbers of RSV CP genes in different tissues were variable. Accumulation of the RSV CP gene was greater in rice leaf and SBPH thoraco-abdominal tissue than in rice stem and SBPH head, respectively. As determined by an end-point dilution comparison, one-step real time RT-PCR was close to 10(4)-fold more sensitive than the indirect enzyme-linked immunosorbent assay (ELISA) for RSV detection. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of RSV biology.