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 10(3)- and 10(5)-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.