A next-generation sequencing approach for the simultaneous detection of plant viruses for plant quarantine testing
Bibliographic record
Abstract
<p>Potato (Solanum tuberosum L.) is one of the most important vegetable crops in the world and plays a vital role in human nutrition and food security. However, potato crops have been known to be infected by at least 40 viruses and 2 viroids contributing to reduced yields. The detection of viruses and viroids is limited from a diagnostic perspective as there are very few validated procedures that exist. Globalization of agriculture has meant that crop plants are now grown further from their centres of origin and far from the pathogens that had co-evolved with them. Crops introduced to a new area may be poorly equipped to resists pathogenic organisms that are already resident. Similarly, increased trade leads to the potential of introducing pathogenic organisms that may not be endemic to a particular region leading to outbreaks having the potential for extreme economic consequences.</p>\n<p>The Potato Post-Entry Quarantine (PPEQ) program implemented by the Canadian Food Inspection Agency (CFIA) was established under the Plant Protection Act to prevent the introduction of economically devastating foreign plant diseases into Canada. The PPEQ program allows the Canadian potato industry to obtain disease-free germplasm from other countries to meet international demands for the production of seed, table, or processing potatoes. The diagnostic methods employed in the current PPEQ program are time-consuming and rely on both traditional and modern techniques. Many of these techniques are only specific for single viral targets having weak specificity and sensitivity. Recently, next-generation sequencing (NGS) platforms have been widely accepted as high-throughput, unbiased technologies that have attractive features in the field of plant diagnostics. NGS has opened the door to very sensitive and specific testing with the opportunity to detect multiple pathogens in a single sample.</p>\n<p>In this study, various RNA extraction methods were evaluated to acquire high quality non-fragmented RNA. Integrity values ranging from 7-9 were obtained using the PureLink Total RNA Mini kit and lysis buffer (Thermo Scientific, Waltham, USA). Using this method for RNA extraction, the application of NGS was explored to determine if plant pathogenic viral genomes could be generated de novo via a bioinformatic pathway when mapped to the whole host genome without a priori knowledge of their presence. Intended potato tuber imports intercepted from Bangladesh showed that viral contigs from multiple mixed infections of Potato aucuba mosaic virus (PAMV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS), Potato virus M (PVM), Potato leaf roll virus (PLRV), and Tomato chlorosis virus (ToCV) could be detected simultaneously with high specificity. Similarly, NGS was successfully applied to determine the causal agents of unknown etiology in tomato (S. lycopersicum) without a priori knowledge of their existence in the sample and confirmed the first report of Southern tomato virus (STV) in Canada. This NGS protocol will aid in the diagnosis of pathogens that were otherwise unable to be tested for within the current PPEQ program as well as identify unknown agents from other samples allowing for the development of new routine diagnostic assays and timely epidemiological and eradication strategies to be performed.</p>\n<p></p>
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".