Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR

Panno, Stefano; Ferriol, Inmaculada; Rangel, Eduardo Espitia; Olmos, Antonio; Han, Chenggui; Martinelli, Federico; Rubio, Luís; Davino, Salvatore

doi:10.1016/j.jviromet.2013.12.002

Cited by 30 publications

(18 citation statements)

References 37 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%

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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%

“…However, these methods could not distinguish the three genotypes of BrYV at a time. Multiplex RT-PCR can be used for simultaneously detection of several viruses in one reaction, and it provides a quick, efficient, reliable and economical way for detection of plant viruses and strains, especially in field samples [7–31]. …”

Section: Introductionmentioning

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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“…M-PCR is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction (López et al 2009). M-PCR is useful because several pathogens frequently infect a single plant Panno et al 2012Panno et al , 2014. The compatibility of the designed primers must be evaluated experimentally.…”

Section: Nucleic Acid-based Methodsmentioning

confidence: 99%

Advanced methods of plant disease detection. A review

Martinelli¹,

Scalenghe²,

Davino³

et al. 2014

Agron. Sustain. Dev.

Self Cite

707

341

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International audiencePlant diseases are responsible for major economic losses in the agricultural industry worldwide. Monitoring plant health and detecting pathogen early are essential to reduce disease spread and facilitate effective management practices. DNA-based and serological methods now provide essential tools for accurate plant disease diagnosis, in addition to the traditional visual scouting for symptoms. Although DNA-based and serological methods have revolutionized plant disease detection, they are not very reliable at asymptomatic stage, especially in case of pathogen with systemic diffusion. They need at least 1–2 days for sample harvest, processing, and analysis. Here, we describe modern methods based on nucleic acid and protein analysis. Then, we review innovative approaches currently under development. Our main findings are the following: (1) novel sensors based on the analysis of host responses, e.g., differential mobility spectrometer and lateral flow devices, deliver instantaneous results and can effectively detect early infections directly in the field; (2) biosensors based on phage display and biophotonics can also detect instantaneously infections although they can be integrated with other systems; and (3) remote sensing techniques coupled with spectroscopy-based methods allow high spatialization of results, these techniques may be very useful as a rapid preliminary identification of primary infections. We explain how these tools will help plant disease management and complement serological and DNA-based methods. While serological and PCR-based methods are the most available and effective to confirm disease diagnosis, volatile and biophotonic sensors provide instantaneous results and may be used to identify infections at asymptomatic stages. Remote sensing technologies will be extremely helpful to greatly spatialize diagnostic results. These innovative techniques represent unprecedented tools to render agriculture more sustainable and safe, avoiding expensive use of pesticides in crop protection

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“…To date, the only two available tools to contain ToBRFV worldwide are early diagnosis and the implementation of preventive measures in crop management, which can be a valuable aid in reducing the introduction and subsequent ToBRFV spread in other countries. Consequently, today there is the need to develop alternative, sensitive and very reliable diagnostic methods for the diagnosis of plant viruses, which are compromising the tomato crops in various Italian and international areas (Hanssen et al, 2010;Puchades et al, 2017;Ferriol et al, 2015, Panno et al, 2014. In the present work a Tomato brown rugose fruit virus isolate, recently found in Sicily (Panno et al, 2019b), was characterised, and a sensitive, specific, rapid and economical method for its detection in infected plants and seeds was developed.…”

Section: Discussionmentioning

confidence: 99%

Peer Review #1 of "Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques (v0.1)"

Alkowni¹

2019

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Background. Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods. Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan MGB probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. Results. The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in Solanum lycopersicum L. and Capsicum annuum L., but no transmission occurred in Solanum melongena L.. The developed Real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the ORF3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, Real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive Real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis,

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Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR

Cited by 30 publications

References 37 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

Advanced methods of plant disease detection. A review

Peer Review #1 of "Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques (v0.1)"

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