Development of a TaqMan real-time RT-PCR protocol for rice stripe necrosis virus detection in plant material and soil

Rice is one of the main staple foodstuffs for the world's population; however, several diseases caused by phytopathogens cause yield losses in rice crops. Rice stripe necrosis virus (RSNV) belonging to the Benyviridae family and Benyvirus oryzae species is the causal agent of rice crinkle, an emerging disease that threatens this important crop. RSNV is transmitted by the plasmodiophoromycete, Polymyxa graminis, which can remain in the soil through resistance structures for decades. The symptoms caused by the virus are often confused with other causes, making a correct diagnosis and the adoption of effective management and control measures difficult. Current RSNV detection methods are mainly based on enzyme-linked immunosorbent assay (PTA-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) assays. In this study, a new detection method based on a TaqMan real-time RT-PCR combined with a direct sample preparation method using crude extracts has been developed. The new protocol has resulted in 100 times more sensitivity than conventional RT-PCR when using plant material and 1000 times more sensitivity for soil solutions. In addition, the high sensitivity exhibited by our method has also been shown when testing direct samples, thus avoiding nucleic acid purification steps.