A One-Step, Real-Time Reverse Transcription Loopmediated Isothermal Amplification Assay to Detect Potato Virus Y

Agnieszka Przewodowska,Bogumiła Zacharzewska,Joanna Chołuj,Krzysztof Treder

doi:10.1007/s12230-015-9430-3

Abstract

Potato viruses such as Potato virus Y (PVY) cause diseases that affect potato quality and thus damage potato production worldwide. Current tests for viral infection use double-antibody sandwich enzyme linked immunosorbent assays (DAS‑ELISA) or reverse transcription polymerase chain reaction (RT-PCR)/real-time RT-PCR. Despite many advantages, these assays have a number of drawbacks that affect cost and time of diagnosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) allows fast detection of target RNA. Here, we developed a closed-tube real-time RT‑LAMP assay for fluorescent detection of PVY. Specific RT-LAMP primers were designed to target the conserved region of the sequence encoding the PVY coat protein. The assay was specific and facilitated sensitive PVY detection in a single tube at 65 °C. The time-to-positive values depended on the PVY concentration in tested samples. The effectiveness of RT‑LAMP in testing field-grown plants compared favorably with DAS‑ELISA and RT-PCR; under the tested conditions, RT-LAMP was about 1000-fold more sensitive than DAS‑ELISA and lateral flow assay (LFA) and about 10-fold more sensitive than RT‑PCR. Thus, this fluorescent RT-LAMP assay has great potential for routine detection of PVY.

Highlights

Materials and MethodsPotato virus Y, a type member of the genus Potyvirus, is considered scientifically and economically as one of the most important viruses distributed worldwide (Quenouille et al 2013; Scholthof et al 2011)
We modified a commercial kit for fluorescent loop-mediated isothermal amplification of DNA (LAMP) amplification by adding thermostable reverse transcriptase (RTase) to the reaction mix
To optimize the reaction conditions, we investigated the influence of the concentration of primers and RTase on the reaction efficacy (Fig. 2)

Summary

Introduction

Potato virus Y, a type member of the genus Potyvirus (family Potyviridae), is considered scientifically and economically as one of the most important viruses distributed worldwide (Quenouille et al 2013; Scholthof et al 2011). PVY causes a major threat to potato yields and significantly affects tobacco, pepper, and tomato (Crosslin 2012; Quenouille et al 2013; Scholthof et al 2011; Singh et al 2008; Wróbel and Wąsik 2013). The most common procedure to detect PVY involves bio-amplification of the virus by cutting eye cores from dormant tubers and growing offspring plants in a greenhouse for 3-4 weeks. The cost and complexity of immunological and molecular methods restricts their use to dedicated, specialized laboratories (Boonham et al 2008)

Methods

Results

Conclusion