@inbook{41692abfb6444c30a4c185879fe0e202,
title = "Rapid and Sensitive Detection of Phytoplasma Diseases Using a CRISPR/Cas12a DETECTR Assay Combined with Isothermal Recombinase Polymerase Amplification",
abstract = "Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal 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 incubation with 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 reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. 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 for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.",
author = "Wei Wei and Yinong Yang and Justin Shih",
note = "Publisher Copyright: {\textcopyright} The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2026.",
year = "2026",
doi = "10.1007/978-1-0716-5104-9\_6",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "63--74",
booktitle = "Methods in Molecular Biology",
}