Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a

<i>Phytophthora cambivora</i> is a major quarantine pathogen that devastates economically important plants across the globe. <i>P. cambivora</i> causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and pl...

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Main Authors: Jing Zhou, Hanqian Dai, Tingting Dai, Tingli Liu
Format: Article
Language:English
Published: MDPI AG 2023-10-01
Series:Forests
Subjects:
Online Access:https://www.mdpi.com/1999-4907/14/11/2141
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author Jing Zhou
Hanqian Dai
Tingting Dai
Tingli Liu
author_facet Jing Zhou
Hanqian Dai
Tingting Dai
Tingli Liu
author_sort Jing Zhou
collection DOAJ
description <i>Phytophthora cambivora</i> is a major quarantine pathogen that devastates economically important plants across the globe. <i>P. cambivora</i> causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and plant death. Given the potential dangers of <i>P. cambivora</i>, effective detection methods are needed for both disease management and prevention. In this study, based on the whole-genome screening of specific target genes, a combination of the recombinase polymerase amplification technique (RPA) and CRISPR/Cas12 was established to detect <i>P. cambivora</i>. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of <i>P. cambivora</i> but did not detect the following 68 non-target isolates, including 28 isolates of <i>Phytophthora</i>, 3 isolates of <i>Pythium</i>, 3 isolates of <i>Phytopythium</i>, 32 isolates of fungi, and 2 isolates of <i>Bursaphelenchus</i>. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL<sup>−1</sup> of <i>P. cambivora</i> genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of <i>P. cambivora</i> in artificially inoculated apple fruits. In summary, compared with conventional detection techniques, the RPA-CRISPR/Cas12a detection method eliminates the need for expensive instruments, long reaction times, and high amounts of raw materials and can detect <i>P. cambivora</i> in imported plants at entry ports, enabling instant prevention and detection.
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spelling doaj.art-fef79e37390a4185a339a01d529e6fe92023-11-24T14:42:28ZengMDPI AGForests1999-49072023-10-011411214110.3390/f14112141Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12aJing Zhou0Hanqian Dai1Tingting Dai2Tingli Liu3Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, ChinaNanjing Institute of Surveying, Mapping & Geotechnical Investigation, Co., Ltd., Nanjing 210005, ChinaCo-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, ChinaSchool of Food Science, Nanjing Xiaozhuang University, 3601 Hongjin Avenue, Nanjing 211171, China<i>Phytophthora cambivora</i> is a major quarantine pathogen that devastates economically important plants across the globe. <i>P. cambivora</i> causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and plant death. Given the potential dangers of <i>P. cambivora</i>, effective detection methods are needed for both disease management and prevention. In this study, based on the whole-genome screening of specific target genes, a combination of the recombinase polymerase amplification technique (RPA) and CRISPR/Cas12 was established to detect <i>P. cambivora</i>. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of <i>P. cambivora</i> but did not detect the following 68 non-target isolates, including 28 isolates of <i>Phytophthora</i>, 3 isolates of <i>Pythium</i>, 3 isolates of <i>Phytopythium</i>, 32 isolates of fungi, and 2 isolates of <i>Bursaphelenchus</i>. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL<sup>−1</sup> of <i>P. cambivora</i> genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of <i>P. cambivora</i> in artificially inoculated apple fruits. In summary, compared with conventional detection techniques, the RPA-CRISPR/Cas12a detection method eliminates the need for expensive instruments, long reaction times, and high amounts of raw materials and can detect <i>P. cambivora</i> in imported plants at entry ports, enabling instant prevention and detection.https://www.mdpi.com/1999-4907/14/11/2141<i>Phytophthora cambivora</i>disease diagnosisrecombinase polymerase amplificationCRISPR/Cas12a
spellingShingle Jing Zhou
Hanqian Dai
Tingting Dai
Tingli Liu
Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
Forests
<i>Phytophthora cambivora</i>
disease diagnosis
recombinase polymerase amplification
CRISPR/Cas12a
title Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
title_full Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
title_fullStr Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
title_full_unstemmed Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
title_short Rapid Detection of <i>Phytophthora cambivora</i> Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a
title_sort rapid detection of i phytophthora cambivora i using recombinase polymerase amplification combined with crispr cas12a
topic <i>Phytophthora cambivora</i>
disease diagnosis
recombinase polymerase amplification
CRISPR/Cas12a
url https://www.mdpi.com/1999-4907/14/11/2141
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