RT-PCR and Real-Time RT-PCR Methods for the Detection of Potato Virus Y in Potato Leaves and Tubers

Abstract

Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications.

Potato virus Y (PVY) detection in a single aphid by one‐step RT‐PCR with boiling technique

“…; Mackenzie et al . ). Whether PVY was inoculated or not is the more important question for seed potato production than what kind of strain was inoculated.…”

“…Several previous PVY detection studies were mainly focused on developing detailed methods such as strain-specific detection or multi-PCR-based techniques (Rigotti & Gugerli 2007;Rupar et al 2013;Mackenzie et al 2015). Whether PVY was inoculated or not is the more important question for seed potato production than what kind of strain was inoculated.…”

“…Recently, various PVY detection methods have been developed from potato leaf and tuber, which have been applied in seed potato cultivation to attempt PVY control (Mackenzie et al . ). PVY displays a large genetic variability and variants linked to a large and variable host range.…”

“…Particularly, several important crop species belonging to the Solanaceae family including potato, Solanum tuberosum, tobacco, Nicotiana tabacum, pepper, Capsicum annuum and tomato, S. lycopersicum, are more prone to PVY, leading to significant yield losses and quality degradation (Scholthof et al 2011). Recently, various PVY detection methods have been developed from potato leaf and tuber, which have been applied in seed potato cultivation to attempt PVY control (Mackenzie et al 2015). PVY displays a large genetic variability and variants linked to a large and variable host range.…”

Potato Cultivar and Seed Type Affect the Development of Systemic Potato virus Y (PVYN-Wi) Infection

“…PVY is a Potyvirus with a 9.7 kb positive sense single-stranded RNA genome and a filamentous capsid (11 nm diameter × 740 nm length) (Scholthof et al 2011). Mutation and recombination have resulted in a great diversity of PVY strains worldwide (MacKenzie et al 2015). The majority of currently circulating PVY strains are recombinant viruses that contain segments of both PVY O and PVY N genomic sequences (Karasev et al 2011).…”