AUTHOR=Guo Yufang , Xia Hongming , Dai Tingting , Liu Tingli , Shamoun Simon Francis , CuiPing Wu 

TITLE=CRISPR/Cas12a-based approaches for efficient and accurate detection of Phytophthora ramorum

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 13 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1218105

DOI=10.3389/fcimb.2023.1218105

ISSN=2235-2988

ABSTRACT=<sec><title>Introduction</title><p><italic>Phytophthora ramorum</italic> is a quarantine pathogen that causes leaf blight and shoot dieback of the crown, bark cankers and death on a number of both ornamental and forest trees, especially in North America and northern Europe, where it has produced severe outbreaks. Symptoms caused by <italic>P. ramorum</italic> can be confused with those by other <italic>Phytophthora</italic> and fungal species. Early and accurate detection of the causal pathogen <italic>P. ramorum</italic> is crucial for effective prevention and control of Sudden Oak Death.</p></sec><sec><title>Methods</title><p>In this study, we developed a <italic>P. ramorum</italic> detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPACRISPR/ Cas12a).</p></sec><sec><title>Results</title><p>This novel method can be utilized for the molecular identification of <italic>P. ramorum</italic> under UV light and readout coming from fluorophores, and can specifically detect <italic>P. ramorum</italic> at DNA concentrations as low as 100 pg within 25 min at 37°C.</p></sec><sec><title>Discussion</title><p>We have developed a simple, rapid, sensitive, unaided-eye visualization, RPA CRISPR/Cas12a-based detection system for the molecular identification of <italic>P. ramorum</italic> that does not require technical expertise or expensive ancillary equipment. And this system is sensitive for both standard laboratory samples and samples from the field.</p></sec>