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

• Published in: Journal of virological methods Vol. 236; pp. 35 - 40
• Main Authors: Kesanakurti, Prasad, Belton, Mark, Saeed, Hanaa, Rast, Heidi, Boyes, Ian, Rott, Michael
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-10-2016
• Subjects: Black Turtle Soup; DsRNA; Mass Screening - methods; Mass Screening - standards; Next-generation sequencing; Nucleic Acid Amplification Techniques - methods; Phaseolus vulgaris; Plant quarantine; Plant viruses; Plant Viruses - genetics; Plant Viruses - isolation & purification; Plants - virology; Reference Standards; RNA, Double-Stranded - genetics; RNA, Double-Stranded - isolation & purification; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sequence Analysis, DNA - methods; Next-generation sequencing; DsRNA; Plant quarantine; Plant viruses; Black Turtle Soup
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2016.07.001

•This method uses 2–5g starting material.•Tissue disruption is achieved through bead based homogenization in closed, disposable, 50ml tubes.•This method employs internal amplification control to monitor the extraction quality and to measure the accuracy of NGS screening for plant viruses.
The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS).