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
Notice bibliographique
Résumé
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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,007 | 0,005 |
| Méta-épidémiologie (sens strict) | 0,002 | 0,001 |
| Méta-épidémiologie (sens large) | 0,001 | 0,001 |
| Bibliométrie | 0,002 | 0,001 |
| Études des sciences et des technologies | 0,001 | 0,001 |
| Communication savante | 0,001 | 0,001 |
| Science ouverte | 0,002 | 0,001 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».