Development of a Real-time RT-PCR Method for Rapid Detection and Quantification of Southern Rice Black-streaked Dwarf Virus in Rice

Abstract

IntroductionSouthern rice black-streaked dwarf virus (SRBSDV), a latest member of the genus Fijivirus within the family Reoviridae, was simultaneously isolated and identified from the rice in southern of China in 2008 [1,2]. In the following three years (2009)(2010), SRBSDV caused the most important rice disease in South of China, there were over ten millions hectare of rice infected, and resulted in massive devastation of rice yield [3].SRBSDV was most closely related to rice black-streaked dwarf virus (RBSDV); symptoms of infected rice by SRBSDV were similar to RBSDV, such as stunting, dark leaf and small enations on stem and leaf back. Its genome consisted of 10 linear segments of double-stranded RNAs (dsRNA) called S1-S10 in an increasing order of electrophoretic mobility in polyacrylamide gels [1]. The homology of nucleotide sequence of genome segments of SRBSDV was 70.6%-80.0% similar to the genome segments of RBSDV. Nevertheless, the SRBSDV was transmitted to rice by white-backed planthoppers, but not by small brown planthopper (RBSDV transmitted vector), and whole genome comparisons and phylogenetic analyses also supported suggestions that SRBSDV represented a new species within the genus Fijivirus, family Reoviridae [4]. So it is exceeding to develop an accurate detecting method for identifying SRBSDV from RBSDV.Currently, the identification of SRBSDV and its infection is mainly by typical symptoms and RT-PCR [5]. For typical symptoms, it is time consuming, false positive in early diagnosis and severe yield loss by the time it is identified in the infected rice [6]. For RT-PCR, thought it is specific to SRBSDV, false positive still constantly occur, especially in early infected stage in rice because of low quantity of virion in the host [7]. The serological methods are more fast and economical than other methods [8] including in rice and transmitted vector, nevertheless, they are under development. These methods are difficult to precisely quantify SRBSDV, and difficult to rapidly detect the disease for early warning [9]. So it is very important to develop a detecting method for SRBSDV with simple operation, strong specificity, high sensitivity, good reproducibility and quick speed. The real-time RT-PCR is suitable and gradually become an important technique for virus detecting [10][11][12].This study aimed to design specific primers and develop an absolute quantification real-time RT-PCR method for detecting SRBSDV, and this method was also applied to detect SRBSDV infection in field samples of rice. For demonstrating the excellent performance of developed real-time RT-PCR, the detection efficiency was also compared with conventional RT-PCR. Materials and Methods Leaf samples of riceLeaf samples of rice were collected from heading stage of rice in different districts of Hunan province, China. The leaf samples were gathered in period from 2009 to 2011, there into, some of samples had typical or suspected symptoms of SRBSDV infected, such as stunting, dark leaf and/or small enations on stem and leaf back....

The detection of avocado sunblotch viroid in avocado using a real-time reverse transcriptase polymerase chain reaction

“…A careful selection of primers has proven to be the most effective approach to increase the sensitivity of the method, compared with RT-PCR (Bustin et al, 2009;Zhang et al, 2013a;Zhang et al, 2013b). When using primers reported by Schnell and coworkers (1997) for RT-qPCR, 4 of the 10 positive samples detected by RT-PCR were reported as negative by the RT-qPCR and further sequencing.…”

Generation of Synthetic Peptide-Specific Antibody for the Development of A Southern Rice Black-Streaked Dwarf Virus Diagnostic Test

“…The P10 protein of SRBSDV is a common target in diagnostic assays of SRBSDV using specific antibodies (Zhang et al, 2013;Do Thi Hanh et al, 2015). To create antibodies specifically binding to the P10 protein of Vietnamese SRBSDV isolates, the peptide fragment antigen was screened according to whether it was located on the (i) antigenic or (ii) conservative region of the P10 protein.…”

“…Currently, the most common SRBSDV diagnostic technique is reverse transcriptionpolymerase chain reaction (RT-PCR) using specific primers designed based on a highly conservative sequence on the S10 segment of the viral genome (Zhang et al, 2008;Ngo Vinh Vien et al, 2009;Cuong et al, 2010;Anh et al, 2011). SRBSDV diagnostic techniques using real-time RT-PCR are also being applied to quantify viruses for early diagnosis (Zhang et al, 2013). Chinese laboratories have produced anti-SRBSDV antibodies using several synthetic peptide fragments of the P10 protein and developed a diagnostic technique using dot-ELISA (Wang et al, 2012).…”

CRISPR/Cas9-mediated gene editing of vacuolar ATPase subunit d mediates phytohormone biosynthesis and virus resistance in rice

“…However, many viral diseases have severely challenged rice production and have increased the risk to food security in many global areas ( Jones and Dangl, 2006 ; Mandadi and Scholthof, 2013 ; Nicaise, 2014 ; Baulcombe, 2019 ; ). For example, Southern rice black-streaked dwarf virus (SRBSDV) is severely epidemic and has caused 30-50% rice yield losses in southern China and Southeast Asia in the last decade ( Hoang et al., 2011 ; Cheng et al., 2013 ; Zhang et al., 2013 ; Zhou et al., 2013 ; ). Rice stripe virus (RSV) is one of the most destructive virus affecting rice production, and it is wide-spread throughout East Asia, especially in China, Japan and Korea ( Hu et al., 2020 ).…”