Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method

Grapevine leafroll disease (GLD) is the most important disease of Grapevines in South Africa. Grapevine leafroll-associated virus type 3 (GLRaV-3) has a close association with the disease and is prevalent in South African vineyards. GLD can be controlled using a combination of virus-free planting material, systemic insecticides to control vector populations and removal of infected vines by roguing. Infected vines are identified each autumn using either symptom display (in red cultivars) or ELISA (in white cultivars). While ELISA is a simple, reliable means of testing for GLRaV-3, it is time consuming, laborious and insensitive and a quicker, more sensitive method of detecting GLRaV-3 in the field is needed. A single-tube one-step reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay combined with a simple RNA extraction protocol was developed for the rapid and easy detection of GLRaV-3. Hydroxy napthol blue was included as an indicator and under isothermal conditions at 60 degrees C the target viral gene could be amplified in under 2 h and positive results could be easily seen by examining the colour change from violet to sky blue. Using this method, 50 samples could be also pooled together with a single positive sample still being detected. A direct comparison of ELISA, nested PCR and RT-LAMP showed that RT-LAMP is as sensitive as nested PCR and could be performed in a much shorter time with less equipment. This assay is may be a possible alternative to ELISA for the detection of GLRaV-3 in the field. (C) 2013 Elsevier B.V. All rights reserved.