Abstract
Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21–24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.
Highlights
The large number and high genetic variability of plant viruses make their detection cumbersome
The RNA concentration was determined using a Nanodrop (Thermo Fischer Scientific, Waltham, MA, United States). 1 μg of leaf RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI, United States) according to the manufacturer’s instructions and used for cDNA synthesis. cDNA synthesis was carried out on 1000 and 250 ng of RNA extracted from purified Potato virus Y (PVY) and Potato virus A (PVA) particles, respectively. cDNA was synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) using random hexamers, according to the manufacturer’s instructions
Total RNA from various crop plants was included in the samples to be analyzed by small RNAs (sRNAs) and RT-RT-polymerase chain reaction (PCR), to maintain the overall amount and concentration of RNA as constant as possible across the samples
Summary
The large number and high genetic variability of plant viruses make their detection cumbersome. SRNAs can be extracted from plant tissues and sequenced, and the sequences can be reassembled into partial or full viral genomes in silico, which allows identification by comparison with viral sequences available in databases. This method, published by Kreuze et al (2009), has been widely adopted and has been used for surveys of viruses in cultivated (Kashif et al, 2012; Moon and Park, 2016; Massart et al, 2017) and wild plants (Bi et al, 2012). It allows the detection of different, unrelated viruses simultaneously in a single assay, without need for antibodies, probes, etc.
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