Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control

Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael

doi:10.1016/j.jviromet.2016.07.001

Cited by 45 publications

(51 citation statements)

References 23 publications

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“…Extraction of dsRNA from grapevine leaves was performed based on a modified version of the technique from Kesanakurti et al [ 28 ]. Differences brought to the protocol were: black bean ( Phaseolus vulgaris , Black Turtle Baking Beans (Vesey, York, PEI, Canada) was added as positive control to the homogenized sample-extraction buffer mix at a final concentration of 1% ( w / w ), and DNase I and RNase T1 digestions were made after the final dsRNA elution.…”

Section: Methodsmentioning

confidence: 99%

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing

Fall

Lemoyne

et al. 2020

Viruses

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Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

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Section: Methodsmentioning

confidence: 99%

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing

Fall

Lemoyne

et al. 2020

Viruses

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“…To address this, two positive control strategies have been developed, using known nucleic acids as an internal control. One uses leaf discs from a plant infected with a known virus (Kesanakurti et al, 2016), whereas the other uses synthetic sequence transcripts (Jiang et al, 2011). Both strategies are considered adequate in current virus discovery applications, however this may not be the case when trying to detect a range of known viruses and other strategies, such as including a suite of non-target viruses as a positive control, could be investigated.…”

Section: Considerations For An Hts Detection Assaymentioning

confidence: 99%

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

et al. 2018

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“…At the same time, its application requires the development of guidelines assuring the reliability and interpretation of the results. Thus far, different approaches have been explored, such as using spikes of endornavirus-infected leaf discs (Kesanakurti et al, 2016) and synthetic sequence transcripts (e.g. For analytical sensitivity some relative and comparative data with conventional methods have been determined (Fox et al, 2015).…”

Section: Concluding Remarks and Discussionmentioning

confidence: 99%

“…For example, it would not be practical to include a positive control for every virus that could possibly be detected in a sample, and use of an internal control seems more appropriate. Thus far, different approaches have been explored, such as using spikes of endornavirus-infected leaf discs (Kesanakurti et al, 2016) and synthetic sequence transcripts (e.g. Jiang et al, 2011).…”

Section: Concluding Remarks and Discussionmentioning

confidence: 99%

Ensuring validation in diagnostic testing is fit for purpose: a view from the plant virology laboratory

Roenhorst¹,

Krom²,

Fox

et al. 2018

EPPO Bulletin

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Plant diagnostic laboratories are facing increasing demands for accreditation of their testing activities from both private and public organizations. For diagnostics, validating tests for the intended use is one of the key elements of accreditation. Thus far, validation has mainly focused on tests with a fixed scope, i.e. a test for organism X in matrix Y by method Z, suitable for the analysis of targeted samples. In many laboratories, however, targeted samples only form a minority of the samples. The majority consists of symptomatic (non‐targeted) samples including a huge variety of pest, host and matrix combinations. Since it is impossible to validate all tests used for the analysis of these non‐targeted samples, ensuring the reliability of the final results needs a different approach. This paper describes such an approach for diagnostic testing in plant virology, where the extent of validation of a test is determined based on the risks of a false result within the context of the diagnostic process. Examples illustrate how this risk‐based system approach relies on a tight inter‐relationship of validation, expertise and quality assurance within the diagnostic laboratory.

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Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control

Cited by 45 publications

References 23 publications

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

Ensuring validation in diagnostic testing is fit for purpose: a view from the plant virology laboratory

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