A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in...

Area of Science:

• Plant virology
• Molecular diagnostics
• Agricultural science

Background:

• Grapevine leafroll disease is caused by Grapevine leafroll-associated viruses (GLRaVs).
• GLRaV-2 is a highly variable species within this complex, posing detection challenges.
• Existing detection methods lack the sensitivity and broad coverage for all GLRaV-2 variants, especially in grapevine rootstocks.

Purpose of the Study:

• To develop a highly sensitive and universally applicable molecular method for detecting all known variants of GLRaV-2.
• To improve the diagnosis of GLRaV-2 infections in grapevines, including rootstocks.
• To facilitate accurate grapevine sanitary selection and disease monitoring.

Main Methods:

• Development of novel universal primers targeting the 3' end of the GLRaV-2 genome.
• Implementation of a one-step SYBR Green real-time RT-PCR assay for quantitative detection.
• Testing the assay with 43 diverse GLRaV-2 isolates from various geographical origins and grapevine varieties.

Main Results:

• The developed real-time RT-PCR assay successfully identified all 43 tested GLRaV-2 isolates reproducibly.
• The new method demonstrated a 125-fold increase in sensitivity compared to conventional single-tube RT-PCR.
• Geographical origin and grapevine variety did not affect the assay's detection capability.

Conclusions:

• The novel real-time RT-PCR assay provides a sensitive, specific, and universal tool for GLRaV-2 detection.
• This method significantly advances grapevine sanitary selection and leafroll disease monitoring capabilities.
• The assay's high sensitivity and broad applicability are crucial for managing GLRaV-2 spread in viticulture.