RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae

Yufang Guo; Hongming Xia; Tingting Dai; Tingli Liu

doi:10.3389/fcimb.2023.1208837

RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae

Front Cell Infect Microbiol. 2023 May 26:13:1208837. doi: 10.3389/fcimb.2023.1208837. eCollection 2023.

Authors

Yufang Guo¹, Hongming Xia¹, Tingting Dai¹, Tingli Liu²

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.

Abstract

Introduction: Phytophthora sojae is among the most devastating pathogens of soybean (Glycine max) and severely impacts soybean production in several countries. The resulting disease can be difficult to diagnose and other Phytophthora species can also infect soybean. Accurate diagnosis is important for management of the disease caused by P. sojae.

Methods: In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system were used for detection of P. sojae. The assay was highly specific to P. sojae.

Results: The test results were positive for 29 isolates of P. sojae, but negative for 64 isolates of 29 Phytophthora species, 7 Phytopythium and Pythium species, 32 fungal species, and 2 Bursaphelenchus species. The method was highly sensitive, detecting as little as 10 pg.µL^-1 of P. sojae genomic DNA at 37°C in 20 min. The test results were visible under UV light and readout coming from fluorophores. In addition, P. sojae was detected from natural inoculated hypocotyls of soybean seedlings using this novel assay. The rapidity and accuracy of the method were verified using 30 soybean rhizosphere samples.

Discussion: In conclusion, the RPA-CRISPR/Cas12a detection assay developed here is sensitive, efficient, and convenient, and has potential for further development as a kit for monitoring root rot of soybean in the field.

Keywords: CRISPR/Cas12a; Phytophthora sojae; detection; disease diagnosis; recombinase polymerase amplification.

Publication types

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

MeSH terms

CRISPR-Cas Systems
Fluorescent Dyes
Nucleotidyltransferases
Phytophthora* / genetics
Recombinases*

Substances

Recombinases
Nucleotidyltransferases
Fluorescent Dyes

Grants and funding

This work was supported by the Jiangsu University Natural Science Research Major Project (21KJA220003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.