Journal of Plant Pathology & Microbiology

Abstract

Visual Detection of Curly Top Virus by the Colorimetric Loop-Mediated Isothermal Amplification

Mohammad Amin Almasi, Mehdi Aghapour-ojaghkandi and Saeedeh Aghaei

Curly Top Virus (CTV) in sugar beet, belonging to the family Geminiviridae, genius Curtovirusis a considerable problem in semiarid areas of the Iran and other semiarid parts of the world. There are several diagnostic methods to detect CTV, But these techniques take a long time for 3 h to tow day, requiring sophisticated tools. The aim of this study, for the first time, was to reduce the time required to detect CTV in sugar beet, using colorimetric loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock.DNA was extracted from infected naturally leaf tissues. Samples were tested for the presence of Curtovirus species by PCR and LAMP reactions to amplify replication-associated protein (rep) gene using species primers. LAMP was optimized to amplify CTV DNA under isothermal conditions by incubation at 63°C for 30 min. LAMP products were detected visually using the different dyes. The LAMP amplification products had a ladder-like appearance when electrophorised on an agarose gel and also positive results using the different dyes were a color change. Results confirmed LAMP with different dyes provides a rapid and safe assay for detection of CTV in sugar beet. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostics in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.