Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput...

Area of Science:

• Plant pathology
• Molecular biology
• Virology

Background:

• Double-stranded RNA (dsRNA) extraction is crucial for detecting RNA viruses, viroids, and some DNA viruses.
• High-throughput sequencing (HTS) coupled with dsRNA enrichment enables broad-spectrum viral detection.
• Existing dsRNA enrichment strategies include cellulose binding, anti-dsRNA antibodies, and viral protein affinity.

Purpose of the Study:

• To comparatively evaluate three dsRNA enrichment methods for virus and viroid detection via HTS.
• To assess the efficiency and suitability of antibody, binding protein, and resin-based dsRNA enrichment techniques.
• To identify the optimal dsRNA enrichment method for grapevine viral diagnostics.

Main Methods:

• Comparative analysis of three dsRNA enrichment methods: anti-dsRNA antibody (mAb rJ2), B2 dsRNA binding protein, and ReliaPrep™ Resin.
• Application of these methods to virus-infected and healthy grapevine test plants.
• High-throughput sequencing (HTS) for analyzing enriched dsRNA extracts.

Main Results:

• All three dsRNA enrichment workflows were comparable, with variations mainly in purification steps.
• The B2 binding protein method yielded a 36× dsRNA enrichment increase, while ReliaPrep™ Resin achieved a 163× increase compared to the mAb rJ2 antibody.
• Both cellulose resin and binding protein methods produced highly enriched dsRNA suitable for HTS.

Conclusions:

• The ReliaPrep™ Resin method demonstrated superior dsRNA enrichment efficiency and cost-effectiveness.
• The consistency and potential for automation make the ReliaPrep™ Resin method ideal for future semi-automated workflows.
• This study provides valuable insights for selecting optimal dsRNA enrichment strategies in plant virology.