|Introducing molecular diagnostic technologies for detecting viruses in potato quarantine testing|
H. XU (1), S. Cody (2), D. L. Hammill (2), X. Li (2). (1) Canadian Food Inspection Agency, Charlottetown Laboratory, Charlettetown, PE, Canada; (2) Canadian Food Inspection Agency, Charlottetown Laboratory, Charlottetown, PE, Canada
In Canada,<i> Solanum</i> germplasm approved for entry under a permit to import must be subjected to a process of quarantine testing in the Potato Post Entry Quarantine (PPEQ) program. The quarantine testing lab in Charlottetown, PE, receives approx. 40 accessions each year. The quarantine testing scheme consists of 3 streams, pre-greenhouse (microplants), greenhouse (grown out plants) and pre-release (microplants) testing. The entire process of quarantine testing takes 8-12 months. Some conventional diagnostic methods, such as bioassay using indicator plants, electron microscopy, ELISA and reverse polyacrylamide gel electrophoresis (R-PAGE) have been employed for many years and still play critical roles in plant quarantine testing. For modernizing this program and improving the efficacy of potato quarantine testing, some molecular diagnostic methods, real-time quantitative PCR (qPCR), RT-PCR, RT-qPCR and LAMP were evaluated and validated in this lab. Known virus isolates were used for evaluating the specificity and sensitivity of the primers and probes. Serial dilutions of RNA extracts and composite samples (w/w, v/v) were used. RT-PCR, LAMP and qPCR were shown to be superior to the conventional technologies in several aspects, such as sensitivity, rapidity and multiplicity. They are at least 128-512 times more sensitive than ELISA and R-PAGE for detecting potato pathogens (several viruses, a viroid) and consume much less time than the bioassay does.