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CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet.

Vanitharani Ramachandran1, John J Weiland1, Melvin D Bolton1

  • 1United States Department of Agriculture, Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND, United States.

Frontiers in Microbiology
|July 26, 2021
PubMed
Summary

A new CRISPR-Cas12a method accurately detects beet necrotic yellow vein virus (BNYVV), the cause of sugarbeet rhizomania. This sensitive diagnostic tool aids growers in managing the disease by identifying BNYVV in infected sugarbeet roots.

Keywords:
BNYVVCRISPRRT-RPArhizomaniasugarbeetvirus

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Area of Science:

  • Plant Pathology
  • Molecular Biology
  • Biotechnology

Background:

  • Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), significantly impacts global sugarbeet yield.
  • Accurate and sensitive detection of BNYVV in plants and soil is crucial for effective disease management and variety selection.

Purpose of the Study:

  • To develop a highly sensitive and specific CRISPR-Cas12a-based detection method for BNYVV in sugarbeet roots.
  • To establish isothermal amplification conditions for viral RNA detection.

Main Methods:

  • Development of a reverse transcription (RT) recombinase polymerase amplification (RPA) assay for isothermal amplification of BNYVV RNA.
  • Design and validation of guide RNA for CRISPR-Cas12a targeting of BNYVV.
  • Sensitivity assessment using serially diluted synthetic BNYVV targets and validation in infected sugarbeet root tissues.

Main Results:

  • The RT-RPA successfully amplified and confirmed BNYVV fragments from sugarbeet roots.
  • The CRISPR-Cas12a system demonstrated high cutting efficiency against BNYVV targets.
  • The developed assay accurately and sensitively detected BNYVV in infected sugarbeet roots, showing specific reporter signals compared to healthy controls.

Conclusions:

  • This study presents the first isothermal RT-RPA and CRISPR-Cas12a-based diagnostic method for detecting BNYVV in sugarbeet roots.
  • The developed CRISPR-Cas12a assay offers a sensitive and specific tool for diagnosing BNYVV, aiding in rhizomania management.