Simultaneous Detection And Differentiation Of Rice Black Streaked Dwarf Virus (RBSDV) And Southern Rice Black Streaked Dwarf Virus (SRBSDV) By Duplex Real Time RT-PCR

In major rice-growing countries, rice viral diseases have been occurring one after another and haveinflicted widespread damage over huge areas. More than15rice viruses, especially planthopper orleafhopper transmitted viruses have reached epidemic proportions in many countries and have causedserious damage in rice. In China, major outbreak of Rice black streaked dwarf virus (RBSDV) wasrecorded in1963. A disease with similar symptoms of RBSDV was first observed in GuangdongProvince, Southern China in2001. The causal agent of the disease was identified as Southern riceblack-streaked dwarf virus (SRBSDV), a tentative species in the genus Fijivirus, family Reoviridae.Both RBSDV and SRBSDV have similar genome structure containing with10segments of dsRNAthat encoded at least six putative structural proteins (P1, P2, P3, P4, P8, and P10) and five putativenonstructural proteins (P6, P7-1, P7-2, P9-1and P9-2). Rice plants infected by either RBSDV orSRBSDV show typical stunting, dark green leaves and small enations on stem and leaf backs. However,sometimes these two viruses can be confused with each other and the disease is also very difficult todistinguish with the diagnostic based on symptoms, because the symptoms of these two diseases mayvary according to the infection of different growth stages. A sensitive, reliable and quantitative methodis required to detect and distinguish for RBSDV and SRBSDV in rice and vector insects.In this study, we developed a sensitive and lineage-specific duplex real time RT-qPCR fordetection of RBSDV and SRBSDV in a single or/and double infection in rice samples. The duplexRT-qPCR was optimized using standard samples transcribed by T7Large Scale RNA ProductionSystem in vitro. We developed a reliable system for duplex RT-qPCR, in which its co-efficiency ofRBSDV and SRBSDV, were91.6%and90.7%, respectively. The coefficient of determination wasmore than0.990; the slope of linear equation was3.542, and3.567, respectively. Out of30samplescollected in North and Central China, which were suspected to be infected with these two viruses,10samples were detected RBSDV positive by RT-PCR and12samples by RT-qPCR. No mixed infectionswere found. Simultaneously, out of total60samples collected from Southern China, which were alsosuspected to be infected with these two viruses,41samples were determined SRBSDV positive byRT-PCR and47samples by RT-qPCR. Also in this case no mixed infections were found. The rice geneseEF-1a and UBQ5were selected as internal controls for quantification assay also performed as goodexpression stability.The duplex RT-qPCR assay provided as a sufficiently sensitive, specific, accurate, reproducibleand rapid tool for the detection and differentiation of RBSDV and SRBSDV. The RT-qPCR assay can beused in routine diagnostic of these two viruses in order to study the disease epidemiology in rice crops...