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

Yan, Shuchen

Dissertation/Thesis

2021

Kibenge, Fred
Xu, Huimin

Martin, Chelsea
Xu, Huimin
Wang, Yingwei
Hale, Larry
Riveroll, Angela

Rodriguez, Juan
Hale, Lawrence
Wang, Yingwei

Master of Science

Pathology and Microbiology

University of Prince Edward Island

Charlottetown, PE

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 Show moreThe 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. In 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. Overall, 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. Show less

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