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

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.

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of <i>Arabidopsis thaliana</i> and model crop plants

“…First, reference genes must show an expression level which is high enough to be quantified easily, or at least without difficulty, in applications, particularly RT-qPCR. To determine a practical threshold to evaluate the ease of quantification, distributions of FPKM values were investigated in 16 genes that have been practically used in RTqPCR as reference genes by researchers (Abiko et al, 2005;Benschop et al, 2007;Papdi et al, 2008;Streitner et al, 2008;Li et al, 2009;Li et al, 2010;Fontaine et al, 2012;Kudo et al, 2012;Zhang et al, 2013;Wang et al, 2014;Yin et al, 2014;Gonzalez-Cabanelas et al, 2015;Kamada-Nobusada et al, 2015;Patil et al, 2015;Zhai et al, 2015). In 15 of the 16 genes, the 25th percentile of the FPKM values was above 10; the exception was the Arabidopsis PPR gene, all of whose FPKM values were below 10 ( Supplementary Fig.…”

A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach

“…Field surveys of peach viruses showed that some peach trees were infected with both ACLSV and CGRMV in China (unpublished data). Recently, three articles have reported plant virus detection using multiple RT-qPCR assays [7-9]. Therefore, we initiated this study to develop a method to determine the absolute copy numbers of ACLSV and CGRMV genomes in peach tissues, and to evaluate a duplex SYBR Green I-based RT-qPCR assay for the detection of ACLSV and CGRMV in a single reaction.…”

Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time

“…Both methods of RT-PCR have been widely used to detect viruses in plants and insect vectors (Lett et al, 2002;Varga and James, 2005;Zhang et al, 2010Zhang et al, , 2013bWatanabe and Bressan, 2013;Wang et al, 2014a,b). In the present study, we sought to quantify two viruses over time in the various organs of their respective vector and nonvector insects using quantitative RT-PCR (RT-qPCR) and conventional RT-PCR.…”