One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses

Cho, Sang-Yun; Jeong, Rae‐Dong; Yoon, Young Mi; Lee, Su-Heon; Shin, Dong Bum; Kang, Hang-Won; Lee, Bong Choon

doi:10.1016/j.jviromet.2013.07.008

Cited by 20 publications

(13 citation statements)

References 22 publications

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“…Particularly, this technique has been used successfully for simultaneous detection and differentiation of the five seedborne legume viruses [35], three ilarviruses affecting stone fruit trees [27], five potato viruses and a viroid [8], six citrus viroids and apple stem grooving virus from citrus plants [36], four viruses and Pseudomonas savastanoi pv. savastanoi in olive trees [10], five tospoviruses in ornamental crops [28], eight stone fruit viruses [37], nine grapevine viruses [12], three beet poleroviruses in sugarbeet and aphids [38, 39], eight wheat viruses [13], nine crinivirus infecting vegetable and small fruit crops [15], four viruses infecting cassava [18], five tobacco viruses [19], three cucurbit-infecting poleroviruses [30], seven main tomato-infecting viruses [20], three rice viruses [21], three lily-infecting viruses [22], four cherry viruses [23], four viruses in peach [24], three viruses in pear plants [40], five viruses and two viriods infecting chrysanthemum [41], three viruses infecting papaya [42], three orchid viruses [25], five fabaviruses [31] and so on.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

Zhang

Peng

Wang

et al. 2016

Virol J

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BackgroundBrassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV.ResultsIn this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China.ConclusionsThe simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

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Section: Discussionmentioning

confidence: 99%

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

Zhang

Peng

Wang

et al. 2016

Virol J

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“…Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using a Multiplex Rice Virus RT-PCR Kit [Intron Biotechnology, Daejeon, Korea (Cho et al 2013)] and the manufacturer's procedure.…”

Section: Rna Extraction and Virus Identificationmentioning

confidence: 99%

A nationwide survey and genetic analysis of rice stripe virus in South Korea during 2012–2013

et al. 2015

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Rice stripe virus (RSV) is a major rice-infecting virus in Korea that has caused a severe resurgence in the western provinces in 2007-2008. The outbreak of RSV in rice was caused by the small brown planthopper Laodelphax striatellus (an RSV vector), which overwinters in temperate climates. Mass immigration of its insect vector from China has been proposed as a critical risk factor in development of the disease. A nationwide survey was done in June and July 2012-2013 in 30 Korean provinces to confirm RSV sequence variation. The sequence of the nucleocapsid protein (NCP) gene and the intergenic region (IR) of RSV RNA3 was analyzed. A phylogenetic analysis of the NCP gene and the IR demonstrated that the 33 isolates were closely related to earlier reported isolates from Japan and China, but the secondary IR structure showed strong geographic clustering. This is the first report on a nationwide RSV survey of Korea. The results will provide information on the relationships among RSV isolates to understand the epidemiology of this rice disease and the effect of climate changes and insect migration.

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“…Deep sequencing and qRT-PCR and are of high sensitivity and specificity [15–17], but expensive. RT-PCR is a rapid, specific and reliable assay to detect RNA viruses [18, 19], especially for viruses that do not have antibodies available.…”

Section: Introductionmentioning

confidence: 99%

Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)

Liu

Yuan

et al. 2017

Virol J

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BackgroundThe small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples.ResultsWe established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation.ConclusionsA simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0732-6) contains supplementary material, which is available to authorized users.

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One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses

Cited by 20 publications

References 22 publications

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

A nationwide survey and genetic analysis of rice stripe virus in South Korea during 2012–2013

Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)

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