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

Abstract

Grapevine leafroll disease (GLD) is the most important disease of Grapevines in South Africa. Grapevineleafroll-associated virus type 3 (GLRaV-3) has a close association with the disease and is prevalent in SouthAfrican vineyards. GLD can be controlled using a combination of virus-free planting material, systemicinsecticides to control vector populations and removal of infected vines by roguing. Infected vines areidentified each autumn using either symptom display (in red cultivars) or ELISA (in white cultivars). WhileELISA is a simple, reliable means of testing for GLRaV-3, it is time consuming, laborious and insensitiveand a quicker, more sensitive method of detecting GLRaV-3 in the field is needed. A single-tube one-stepreverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay combined with a simpleRNA extraction protocol was developed for the rapid and easy detection of GLRaV-3. Hydroxy naptholblue was included as an indicator and under isothermal conditions at 60◦C the target viral gene couldbe amplified in under 2 h and positive results could be easily seen by examining the colour change fromviolet to sky blue. Using this method, 50 samples could be also pooled together with a single positivesample still being detected. A direct comparison of ELISA, nested PCR and RT-LAMP showed that RT-LAMPis as sensitive as nested PCR and could be performed in a much shorter time with less equipment. Thisassay is may be a possible alternative to ELISA for the detection of GLRaV-3 in the field.