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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Publications at this Location » Publication #413986
Research Project: Development of Applied Management Systems for Diseases of Perennial Crops with Emphasis on Vector-Borne Pathogens of Grapevine and Citrus

Location: Crop Diseases, Pests and Genetics Research

Title: Development of a CRISPR/SHERLOCK-based method for rapid and sensitive detection of selected pospiviroids

Author
item Zhai, Ying
item GNANASEKARAN, PRABU - Washington State University
item PAPPU, HANU - Washington State University

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2024
Publication Date: 7/4/2024
Citation: Zhai, Y., Gnanasekaran, P., Pappu, H.R. 2024. Development of a CRISPR/SHERLOCK-based method for rapid and sensitive detection of selected pospiviroids. Viruses. 16(7). Article 1079. https://doi.org/10.3390/v16071079.
DOI: https://doi.org/10.3390/v16071079

Interpretive Summary: Viroids are single-stranded, circular RNAs molecules that infect mainly plants. Pospiviroid is a economically important genus that can infect a wide range of plant species. Most pospiviroids can be transmitted to potato and tomato, with similar symptoms being observed under controlled conditions. In addition to mechanical transmission, several pospiviroids have been reported to be transmitted via pollen and seeds. Pospiviroids continue to be a major concern of production constraint as well as a quarantine reason for new germplasm arriving in the United States. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance, or produced in countries where these pospiviroids are not known to occur. The six pospiviroids of quarantine interest include potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato planta machoviroid (TPMVd), pepper chat fruit viroid (PCFVd), columnea latent viroid (CLVd), tomato apical stunt viroid (TASVd). Currently, those six viroids are detected by real time quantitative PCR. In this research, we developed a rapid SHERLOCK platform for pospiviroid detection. It’s fast, does not require expensive equipment, and can be adapted for on-site detection.

Technical Abstract: Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.