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.

Peng Zhang,Xifeng Wang,Li Li,Thi Thi Mar,Wenwen Liu

doi:10.1186/1743-422x-10-24

Abstract

BackgroundThe diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years. A sensitive, reliable and quantitative method is required to detect and distinguish for RBSDV and SRBSDV in rice and vector insects.ResultsWe developed a sensitive and lineage-specific duplex real time RT-qPCR for detection of RBSDV and SRBSDV in a single or/and double infection in rice samples. The duplex RT-qPCR was optimized using standard samples transcribed by T7 Large Scale RNA Production System in vitro. We developed a reliable system for duplex RT-qPCR, in which its co-efficiency of RBSDV and SRBSDV, were 91.6% and 90.7%, respectively. The coefficient of determination was more than 0.990; the slope of linear equation was −3.542, and −3.567, respectively. Out of 30 samples collected in North and Central China, which were suspected to be infected with these two viruses, 10 samples were detected RBSDV positive by RT-PCR and 12 samples by RT-qPCR. No mixed infections were found. Simultaneously, out of total 60 samples collected from Southern China, which were also suspected to be infected with these two viruses, 41 samples were determined SRBSDV positive by RT-PCR and 47 samples by RT-qPCR. Also in this case no mixed infections were found. The rice genes eEF-1a and UBQ5 were selected as internal controls for quantification assay also performed as good expression stability.ConclusionThe duplex RT-qPCR assay provided as a sufficiently sensitive, specific, accurate, reproducible and rapid tool for the detection and differentiation of RBSDV and SRBSDV. The RT-qPCR assay can be used in routine diagnostic of these two viruses in order to study the disease epidemiology in rice crops.

Highlights

The diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years
Standard curves of RT-qPCR The linear range of quantification of the one-step RT-qPCR assay for RBSDV was determined by using tenfold serial dilutions of the standard ssRNA ranging from 10 to 1 × 105 copies to determine the end-point limit of detection and the linearity of the assay (Figure 1A)
Standard curves of duplex RT-qPCR The linear range of quantification of the one-step duplex RT-qPCR assay for segment RNA4 of RBSDV and SRBSDV were determined by using ten-fold serial dilutions of the mixed standard ssRNA as template ranging from 10 to 1 × 105 copies to determine the end-point limit of detection and the linearity of the assay (Figure 3)

Summary

Introduction

The diseases caused by Rice black streaked dwarf virus (RBSDV) and Southern rice black streaked dwarf virus (SRBSDV) have been occurring epidemically in China and southeastern Asia in recent years. More than 15 rice viruses, especially planthopper or leafhopper transmitted viruses have reached epidemic proportions in many countries and have caused serious damage in rice [1]. In China, major outbreak of Rice black streaked dwarf virus (RBSDV) was recorded after 1963, when double-cropping of rice became countries in Southeastern Asia, including Vietnam and Thailand [7,8]. RBSDV occurs mainly in China, Japan and Korea [1]. It is a member of the genus Fijivirus within the family Reoviridae [9,10]. Maize (Zea mays), barnyard grass (Echinochloa crusgalli), flaccid grass (Pennisetum flaccidum) and Juncellus serotinus are the hosts of SRBSDV [6,14]

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Discussion

Conclusion