Biologie moléculaire : accès libre

Establishment and Application of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Detection of Grapevine Fanleaf Virus

Abstract

Mohammadamin Almasi

Grapevine degeneration in grapevines caused by Grapevine Fanleaf Virus (GFLV) has been documented in many viticulture regions worldwide. It is one of the major economically important virus diseases affecting the longevity of grapevines and reducing the fruit yield and fruit quality. To diminish the time required for some diagnostic assays, including reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and also double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture RT-PCR (IC-RT-PCR) protocol on the basis of GFLV genome were used and the reaction conditions were optimized for rapid detection of GFLV. In this regard, DAS-ELISA was employed first to validate the existence of the virus. All six RT-LAMP primers (i.e., F3, B3, FIP, BIP, LF and LB) together with RT-PCR primers (F and B) were designed on the basis of coat protein (CP) gene of GFLV. LAMP method, on the whole, had the following advantages over the other mentioned procedures: (I) fascinatingly, no need of RNA extraction; (II) no requirement of expensive and sophisticated tools for amplification and detection; (III) no post-amplification treatment of the amplicons; and (IV) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes.