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ORIGINAL RESEARCH article

Front. Microbiol.
Sec. Microbe and Virus Interactions with Plants
Volume 15 - 2024 | doi: 10.3389/fmicb.2024.1390422

Cas-OPRAD: A One-pot RPA/PCR CRISPR/Cas12 Assay for On-site Phytophthora root rot Detection

Provisionally accepted
Zhiting Li Zhiting Li 1*Wanzhen Feng Wanzhen Feng 1*Zhaobing Zhu Zhaobing Zhu 2*Shengdan Lu Shengdan Lu 1*Min-Ze Lin Min-Ze Lin 1*Jiali Dong Jiali Dong 3*Zhixin Wang Zhixin Wang 1*Fuxiu Liu Fuxiu Liu 4*Qinghe Chen Qinghe Chen 1*
  • 1 School of Breeding and Multiplication, Hainan University, Haikou, Hainan Province, China
  • 2 School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, Shanghai Municipality, China
  • 3 China Agricultural University, Beijing, Beijing Municipality, China
  • 4 Post-Entry Quarantine Center for Tropical Plant, Haikou Customs District, Haikou, China

The final, formatted version of the article will be published soon.

    Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora 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 Phytophthora root rot (P. sojae). 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 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg μL -1 , while the RPA-Cas12a assay achieved a detection limit of 10 pg μL -1 . Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (< 1 hour), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.

    Keywords: Phytophthora sojae, One-pot RPA/PCR, Recombinase polymerase amplification (rpa), CRISPR/Cas12a, on-site detection, visual detection

    Received: 23 Feb 2024; Accepted: 21 May 2024.

    Copyright: © 2024 Li, Feng, Zhu, Lu, Lin, Dong, Wang, Liu and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Zhiting Li, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China
    Wanzhen Feng, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China
    Zhaobing Zhu, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, Shanghai Municipality, China
    Shengdan Lu, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China
    Min-Ze Lin, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China
    Jiali Dong, China Agricultural University, Beijing, 100083, Beijing Municipality, China
    Zhixin Wang, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China
    Fuxiu Liu, Post-Entry Quarantine Center for Tropical Plant, Haikou Customs District, Haikou, China
    Qinghe Chen, School of Breeding and Multiplication, Hainan University, Haikou, 570228, Hainan Province, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.