Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR

Malandraki, Ioanna; Isaioglou, Ioannis; Olmos, Antonio; Varveri, C.; Vassilakos, Nikon

doi:10.1371/journal.pone.0180877

Cited by 21 publications

(21 citation statements)

References 34 publications

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“…The use of the highly sensitive qPCR arises as an extremely useful tool for studying various agents of infectious. In plant pathology, this technology is increasingly being used for studying various causal agents of plant diseases, including viruses (Poojari et al, 2016; Malandraki et al, 2017; Abdullah et al, 2018; Baaijens et al, 2018; Campos et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…The advancements of the molecular virology and biotechnology have witnessed major breakthroughs in the recent years resulting in highly sensitive and effective technologies/methods (Yadav and Khurana, 2016). In plant virology, real-time quantitative PCR (qPCR) is increasingly being used to improve sensitivity and accuracy while maintaining reliability (Poojari et al, 2016; Malandraki et al, 2017). The use of qPCR instrumentation presents several advantages, since it requires considerably short hands-on time, and detection of amplified products is automated, simple, and reproducible (Espy et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

et al. 2019

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Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative effects on olive oil quality. The work presented here describes the development and application of a new SYBR ® Green-based real-time quantitative PCR (qPCR) analysis for specific and reliable quantification of highly spread olive tree viruses: Olive latent virus 1 (OLV-1) , Tobacco necrosis virus D (TNV-D), Olive mild mosaic virus (OMMV), and Olive leaf yellowing-associated virus (OLYaV). qPCR methodology revealed high specificity and sensitivity, estimated in the range of 0.8–8 copies of the virus genome, for the studied viruses. For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. “Galega vulgar” from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR ® Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

et al. 2019

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“…Multiplex PCR or RT-PCR have been used to identify: i) the main viruses infecting a particular crop, such as tomato, tobacco, legumes, potato, ornamentals, cucumber and olive ( Bariana et al., 1994 ; Bertolini et al., 2001 ; Dai et al., 2012 ; Panno et al., 2012 ; Ge et al., 2013 ; Ali et al., 2014 ; Pallás et al., 2018 ); ii) viruses from the same genus ( Uga and Tsuda, 2005 ; Hu et al., 2010 ; Panno et al., 2014 ); and iii) different strains of a viral species ( Hammond et al., 1999 ; Nie and Singh, 2003 ; Huang et al., 2004 ; Alfaro-Fernández et al., 2009 ; Bester et al., 2012 ). Multiplex real-time qPCR with Taqman probes labeled with different fluorescent dyes have been used to identify viruses from the same crop ( Abrahamian et al., 2013 ; López-Fabuel et al., 2013 ; Malandraki et al., 2017 ), and strains or isolates from the same viral species ( Varga and James, 2005 ; Debreczeni et al., 2011 ). The main problem of multiplex PCR is that only a limited number of targets can be amplified simultaneously since the more primers are used, the higher is the probability of incompatibility between some of them.…”

Section: Detection Of Plant Virusesmentioning

confidence: 99%

Detection of Plant Viruses and Disease Management: Relevance of Genetic Diversity and Evolution

2020

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Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. The frequent emergence of new viral diseases is mainly due to international trade, climate change, and the ability of viruses for rapid evolution. Disease control is based on two strategies: i) immunization (genetic resistance obtained by plant breeding, plant transformation, cross-protection, or others), and ii) prophylaxis to restrain virus dispersion (using quarantine, certification, removal of infected plants, control of natural vectors, or other procedures). Disease management relies strongly on a fast and accurate identification of the causal agent. For known viruses, diagnosis consists in assigning a virus infecting a plant sample to a group of viruses sharing common characteristics, which is usually referred to as species. However, the specificity of diagnosis can also reach higher taxonomic levels, as genus or family, or lower levels, as strain or variant. Diagnostic procedures must be optimized for accuracy by detecting the maximum number of members within the group (sensitivity as the true positive rate) and distinguishing them from outgroup viruses (specificity as the true negative rate). This requires information on the genetic relationships within-group and with members of other groups. The influence of the genetic diversity of virus populations in diagnosis and disease management is well documented, but information on how to integrate the genetic diversity in the detection methods is still scarce. Here we review the techniques used for plant virus diagnosis and disease control, including characteristics such as accuracy, detection level, multiplexing, quantification, portability, and designability. The effect of genetic diversity and evolution of plant viruses in the design and performance of some detection and disease control techniques are also discussed. High-throughput or next-generation sequencing provides broad-spectrum and accurate identification of viruses enabling multiplex detection, quantification, and the discovery of new viruses. Likely, this technique will be the future standard in diagnostics as its cost will be dropping and becoming more affordable.

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“…Generally, detection of viral pathogens by genome amplification techniques (Polymerase chain reaction: PCR, Reverse transcription polymerase chain reaction: RT-PCR and Loop-mediated isothermal amplification: LAMP) is more sensitive than ELISA, while offering the possibility of multiple detection [ 22 , 23 , 24 , 25 ]. However, the development of specific molecular tests is strongly dependent on the knowledge of the pathogen genome and its molecular diversity, making primer design highly dependent on (or limited by) available sequence datasets.…”

Section: Introductionmentioning

confidence: 99%

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies

Maliogka

Minafra

Saldarelli

et al. 2018

Viruses

Self Cite

105

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Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

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Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR

Cited by 21 publications

References 34 publications

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

Detection of Plant Viruses and Disease Management: Relevance of Genetic Diversity and Evolution

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies

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