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

Yufang Guo; Hongming Xia; Tingting Dai; Tingli Liu; Simon Francis Shamoun; Wu CuiPing

doi:10.3389/fcimb.2023.1218105

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

Front Cell Infect Microbiol. 2023 Jun 27:13:1218105. doi: 10.3389/fcimb.2023.1218105. eCollection 2023.

Authors

Yufang Guo¹, Hongming Xia¹, Tingting Dai¹, Tingli Liu², Simon Francis Shamoun³, Wu CuiPing⁴

Affiliations

¹ Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu, China.
² Jiangsu Provincial Key Construction Laboratory of Special Biomass Resource Utilization, Nanjing Xiaozhuang University, Nanjing, China.
³ Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria, BC, Canada.
⁴ Animal, Plant and Food Inspection Center, Nanjing Customs, Nanjing, Jiangsu, China.

Abstract

Introduction: Phytophthora ramorum 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 P. ramorum can be confused with those by other Phytophthora and fungal species. Early and accurate detection of the causal pathogen P. ramorum is crucial for effective prevention and control of Sudden Oak Death.

Methods: In this study, we developed a P. ramorum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPACRISPR/ Cas12a).

Results: This novel method can be utilized for the molecular identification of P. ramorum under UV light and readout coming from fluorophores, and can specifically detect P. ramorum at DNA concentrations as low as 100 pg within 25 min at 37°C.

Discussion: We have developed a simple, rapid, sensitive, unaided-eye visualization, RPA CRISPR/Cas12a-based detection system for the molecular identification of P. ramorum 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.

Keywords: CRISPR/Cas12a; Phytophthora ramorum; disease diagnosis; plant destroyers; rapid detection.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
DNA
Europe
North America
Phytophthora* / genetics

Substances

DNA

Grants and funding

This work was supported by the National Key R&D Program of China (2021YFD1400100, 2021YFD1400103), the Natural Science Foundation of Jiangsu Province, China (BK 20191389), the Jiangsu University Natural Science Research Major Project (21KJA220003), the Qinglan Project of 2020, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.