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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #418974
Research Project: Omics-Based Approach to Detection, Identification, and Systematics of Plant Pathogenic Phytoplasmas and Spiroplasmas

Location: Molecular Plant Pathology Laboratory

Title: Rapid and sensitive detection of phytoplasma diseases using a CRISPR/Cas12a DETECTR assay combined with RPA

Author
item Wei, Wei
item YANG, YINONG - Pennsylvania State University
item SHIH, JUSTIN - Orise Fellow

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 8/25/2025
Publication Date: 1/14/2026
Citation: Wei, W., Yang, Y., Shih, J. 2026. Rapid and sensitive detection of phytoplasma diseases using a CRISPR/Cas12a DETECTR assay combined with RPA. In: Janik, K. Tabarelli, M. editors. Phytoplasma: Methods and Protocols. New York, NY: Springer US. 3008:63-74. https://doi.org/10.1007/978-1-0716-5104-9_6.
DOI: https://doi.org/10.1007/978-1-0716-5104-9_6

Interpretive Summary:

Technical Abstract: Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporter, generating a fluorescent signal for sensitive pathogen detection. The protocol provides detailed instructions on (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed to help researchers and agricultural professionals effectively adopt and implement this advanced diagnostic technique.