AUTHOR=Li Zhiting , Feng Wanzhen , Zhu Zaobing , Lu Shengdan , Lin Mingze , Dong Jiali , Wang Zhixin , Liu Fuxiu , Chen Qinghe 

TITLE=Cas-OPRAD: a one-pot RPA/PCR CRISPR/Cas12 assay for on-site Phytophthora root rot detection

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1390422

DOI=10.3389/fmicb.2024.1390422

ISSN=1664-302X

ABSTRACT=<p><italic>Phytophthora sojae</italic> is a devastating plant pathogen that causes soybean <italic>Phytophthora</italic> root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of <italic>Phytophthora</italic> root rot (<italic>P. sojae</italic>). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 <italic>P. sojae</italic> from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. μL<sup>−1</sup>, while the RPA-Cas12a assay achieved a detection limit of 10 pg. μL<sup>−1</sup>. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of <italic>P. sojae</italic> on the infected field soybean samples. This assay provides a simple, efficient, rapid (&lt;1 h), and visual detection platform for diagnosing <italic>Phytophthora</italic> root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of <italic>Phytophthora</italic> root rot in the field or customs fields.</p>