Development of SYBR Green I based real-time PCR assays for quantitative detection of Rice tungro bacilliform virus and Rice tungro spherical virus

Rice tungro disease, caused by simultaneous infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), is an important cause of reduced rice harvests in South and Southeast Asia. Although various biological, serological and molecular techniques have been reported previously for the detection of RTBV and RTSV, a method that determines accurately the exact viral load in a tungro affected plant is still not available. The present study describes a method for the absolute quantitation of RTBV and RTSV using SYBR Green I based real-time PCR. The number of copies of RTBV DNA and RTSV RNA present in a tungro affected rice plant at two different time points after inoculation was determined. The sensitivity of real-time PCR based detection was found 103- and 105-folds higher than dot-blot hybridization and standard PCR assays respectively. In addition, the method was used for the simultaneous detection of RTBV and RTSV in a single reaction on the basis of melt curve analysis.

► The real-time PCR was standardized for detection and quantitation of RTBV and RTSV.
► The number of copies of RTBV and RTSV in a tungro affected rice plant was estimated.
► Both viruses were detected simultaneously by a duplex real-time RT-PCR.
► The real-time PCR was more sensitive than dot-blot hybridization and PCR assays.