Visual Detection of Potato leafroll virus by One‐step Reverse Transcription Loop‐Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

Abstract

Loop‐mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) to visually detect Potato leafroll virus. One‐step RT‐LAMP was performed using RNA of PLRV‐infected potato leaves and a set of primers (F3, B3, FIP, BIP, LF and LB) designed for RT‐LAMP reaction of the coat protein (CP) gene of PLRV. Positive effects of RT‐LAMP were detected by agarose gel electrophoresis and hydroxynaphthol blue (HNB) dye and were shown by a colour change from violet to sky blue. RT‐LAMP with HNB dye proved to be a simple assay for the rapid detection of PLRV.

Detection of Coat Protein Gene of the Potato Leafroll Virus by Reverse Transcription Loop-Mediated Isothermal Amplification

“…Since there is no effective viricide to control of the viruses in potato, using virus free seed potatoes is an effective approach for management of potato viral disease. In Iranian seed potato market, along with other part of the world, demands for certified potato seed tubers are increasing [2][3]30]. In order to produce infection free potato seed tubers, using rapid, cost-effective and sensitive methods for detection of important potato viruses, including PLRV, in different stages of infection process would provide a great step forward.…”

“…Up to now, numerous studies in both plants and animals have been reported to recognize pathogens by LAMP technique. For instance, RT-LAMP method were utilized for detection of Plasmodium falciparum gametocytes [9], Fusarium graminearum [23], Peste des petits ruminants virus (PPRV) [24], Potato virus Y (PVY) [10], Japanese yam mosaic potyvirus (JYMV) [22], Rabies virus (RABV) [19], Macrobrachium rosenbergii noda virus (MrNV) [25], Cymbidium *Corresponding author: Mohammad Amin Almasi, Department of Plant Biotechnology, Faculty of Agriculture, University of Zanjan, Zanjan, Iran, E-mail: aminalmasi66@gmail.com mosaic virus (CymMV) [26] and Potato Leafroll Virus (PLRV) [2,3]. As described earlier, some different visualizing systems are employed for verification of LAMP products.…”

“…Additionally, PLRV can be transmitted in a persistent manner by a limited number of aphid species [12]. Also, the two main vector aphids are the green peach aphid (Myzus persicae) and the potato aphid (Macrosiphum euphorbiae) [2,9]. PLRV has isometric capsids containing a singlestranded (ss) messenger-sense RNA (5.9 kb) molecule with several open reading frames (ORFs) linking to a small protein (VPg) [13,14].…”

“…To address these limitations, isothermal-based detection methods such as loop-mediated isothermal amplification (LAMP) were developed. Some different form of this technique including RT-LAMP and IC-RT-LAMP were universally used [2,3,20,21]. Briefly, the LAMP assay can specifically amplified DNA sequences by using a set of 4 primers, which recognize 6 distinct regions on the target DNA.…”

“…Despite the presence of a broad spectrum of diverse deleterious viruses, the most common and economic ones are Potato virus A, Potato virus Y (PVA, PVY, both from genus Potyviruses), Potato leafroll virus (PLRV, genus Polerovirus), Potato virus X (PVX, genus Potexvirus) and Potato virus S (PVS, genus Carlavirus) [1][2][3][4]. Viruses of the family Luteoviridae belongs to ssRNA plant viruses, divided into three genera: Enamovirus, Luteovirus and Polerovirus [5].…”