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

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.