Analytical sensitivity of molecular techniques used for the detection of grapevine leafroll virus 3 (GLRaV-3), a comparative evaluation

Grapevine leafroll disease (GLD) causes a substantial economic impact, with estimated global yield reductions of around 50%. GLD impairs the vines’ general growth and vigor and decreases berries’ quality. GLD is caused by grapevine leafroll-associated viruses (GLRaVs), GLRaV-3 being the most destructive and globally widespread. Sensitive and accurate diagnostics of GLRaV-3 prevent the introduction of infected vines to new vineyards and help replace the infected ones. Therefore, the effectiveness of different molecular diagnostic techniques for GLRaV-3 detection should be evaluated. This work compared the analytical sensitivity (AS) of loop-mediated amplification (LAMP), end-point PCR (epPCR), real-time quantitative PCR (qPCR), and digital PCR (dPCR) for diagnosing GLRaV-3. The most sensitive techniques were dPCR and LAMP. Both methods require extreme care in their execution to avoid contamination. The AS results of the epPCR and qPCR techniques were 1,000 and 10,000 viral genome equivalents, respectively; qPCR had a good dynamic range and linearity (up to 5 dilutions) that covered samples with high viral titer, while dPCR resulted in a better dynamic range (7 dilution points) that covered lower viral copy numbers. This work helps similar models where a reliable technique should be optimized to detect plant viruses according to available resources.