Development and validation of multiplex polymerase chain reaction based diagnostic procedures for detecting eight viruses and one viroid in the Potato Nuclear Stock Certification Program
Bibliographic record
Abstract
The Canadian Food Inspection Agency implements the Nuclear Stock Certification Program to ensure the production of high-quality and disease-free seed potatoes. Currently, this program employs double and triple antibody sandwich enzyme-linked immunosorbent assays (DAS- or TAS-ELISA) and return-polyacrylamide gel electrophoresis (R-PAGE) for detecting the targeted viruses and PSTVd (Potato spindle tuber viroid), respectively. Pathogen-specific antibodies for ELISA are commercially available for detecting PVM (Potato virus M), PMTV (Potato mop-top virus), PotLV (Potato latent virus), PLRV (Potato leafroll virus), PVY (Potato virus Y), PVS (Potato virus S), PVA (Potato virus A), and PVX (Potato virus X). However, the aforementioned methods are highly labour-intensive and time-consuming procedures with low sensitivity that require large sample inputs for screening. Moreover, both methods are involved with extensive experimental procedures and only allow for the screening of one pathogen each time, which further prolongs and complicates the overall diagnosing process.\nIn this study, multiplex PCR-based procedures were developed and validated to index the potato nuclear stock materials simultaneously in a qualitative way (“positive” or “negative”). Two mxRT-PCR (multiplex RT-PCR) and three mxRT-qPCR (multiplex real-time quantitative RT-PCR) procedures were developed for primary screening and confirmatory testing of all targeted pathogens under this program, respectively. As little as 1.0 μl of tissue sap from a microplant (in vitro plantlet) or 1.0 pg of total RNA extract was sufficient for reliable RT-PCR detection. Additionally, virus-specific RNA could be detected in RNA extracts with almost no non-specific amplification in the presence of other viral RNAs. The use of potato genome-specific primer set as an internal control provided a reference for assessing the quality of the RNA extracts and the amplification of the targeted RNAs. Furthermore, the genomes of virus and viroid isolates or strains used in this research were verified using the next-generation sequencing (NGS) technology to ensure the accuracy of the RT-PCR primers employed. All tests conducted using the mxRT-PCR and mxRT-qPCR procedures were validated using another diagnostic assay and known/unknown microplants.\nOverall, the developed and validated standard multiplex PCR-based procedures in this study profoundly improved the sensitivity, specificity, efficiency, and feasibility with reductions in time and labour expenses for routine diagnostic testing of the nine targeted pathogens under the Nuclear Stock Certification Program compared to that of the currently employed ELISA and R-PAGE methods. Subsequently, the multiplex RCR-based procedures involved in this study are ready for implementation into the Nuclear Stock Certification Program. With the accurate and reliable diagnostic results generated by the multiplex PCR-based procedures, CFIA’s reputation and credibility for certifying potato nuclear stock materials will be further enhanced. Ultimately, this study promotes the trade of potato nuclear stock materials and collaboration between CFIA and the potato industries.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.007 | 0.005 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".