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Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput

Michael E Rott1, Kankana Ghoshal1, Sylvain Lerat2

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Journal of Virological Methods
|July 26, 2024
PubMed
Summary

This study compares three double-stranded RNA (dsRNA) enrichment methods for virus detection using high-throughput sequencing (HTS). The ReliaPrep™ Resin method offers the most cost-effective and consistent dsRNA enrichment for HTS applications.

Keywords:
Anti-dsRNA antibodyDsRNAFlock house virus B2Next generation sequencingPlant virusResin

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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.