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Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of gr...

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Autores principales: Zhou, Qinzheng, Liu, Ya, Wang, Zheng, Wang, Huimin, Zhang, Xingyao, Lu, Quan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8971978/
https://www.ncbi.nlm.nih.gov/pubmed/35371187
http://dx.doi.org/10.3389/fpls.2022.856109
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author Zhou, Qinzheng
Liu, Ya
Wang, Zheng
Wang, Huimin
Zhang, Xingyao
Lu, Quan
author_facet Zhou, Qinzheng
Liu, Ya
Wang, Zheng
Wang, Huimin
Zhang, Xingyao
Lu, Quan
author_sort Zhou, Qinzheng
collection PubMed
description The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of great significance. Traditional molecular diagnostic methods are time-consuming and require sophisticated instruments or skilled operators, which are unavailable in resource-limited settings. A specific, sensitive, and field-applicable diagnostic method is urgently needed. In this study, we developed a diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus. The false-positive signals from primer-dependent artifacts were eliminated using a probe, and base substitutions were included in the primer and probe. The entire detection process for the RPA-LFD assay can be completed under 38°C within approximately 30 min, including 15 min for crude nematode genomic DNA (gDNA) extraction and master mix preparation, 15 min for the RPA-LFD assay. This assay displayed high specificity toward B. xylophilus and showed no cross-reactions with closely related species, including Bursaphelenchus mucronatus and Bursaphelenchus doui. The sensitivity of this assay had a detection limit as low as 1 pg of B. xylophilus purified genomic DNA. Furthermore, the application of the RPA-LFD assay in simulated spiked pinewood samples showed accurate detection results. The RPA-LFD assay in this study successfully detected B. xylophilus in less than 30 min, providing a novel alternative for the simple, sensitive, and specific detection of B. xylophilus and showed potential for B. xylophilus point-of-care testing (POCT) in resource-limited areas or in field.
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spelling pubmed-89719782022-04-02 Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts Zhou, Qinzheng Liu, Ya Wang, Zheng Wang, Huimin Zhang, Xingyao Lu, Quan Front Plant Sci Plant Science The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of great significance. Traditional molecular diagnostic methods are time-consuming and require sophisticated instruments or skilled operators, which are unavailable in resource-limited settings. A specific, sensitive, and field-applicable diagnostic method is urgently needed. In this study, we developed a diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus. The false-positive signals from primer-dependent artifacts were eliminated using a probe, and base substitutions were included in the primer and probe. The entire detection process for the RPA-LFD assay can be completed under 38°C within approximately 30 min, including 15 min for crude nematode genomic DNA (gDNA) extraction and master mix preparation, 15 min for the RPA-LFD assay. This assay displayed high specificity toward B. xylophilus and showed no cross-reactions with closely related species, including Bursaphelenchus mucronatus and Bursaphelenchus doui. The sensitivity of this assay had a detection limit as low as 1 pg of B. xylophilus purified genomic DNA. Furthermore, the application of the RPA-LFD assay in simulated spiked pinewood samples showed accurate detection results. The RPA-LFD assay in this study successfully detected B. xylophilus in less than 30 min, providing a novel alternative for the simple, sensitive, and specific detection of B. xylophilus and showed potential for B. xylophilus point-of-care testing (POCT) in resource-limited areas or in field. Frontiers Media S.A. 2022-03-18 /pmc/articles/PMC8971978/ /pubmed/35371187 http://dx.doi.org/10.3389/fpls.2022.856109 Text en Copyright © 2022 Zhou, Liu, Wang, Wang, Zhang and Lu. https://creativecommons.org/licenses/by/4.0/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) and the copyright owner(s) 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.
spellingShingle Plant Science
Zhou, Qinzheng
Liu, Ya
Wang, Zheng
Wang, Huimin
Zhang, Xingyao
Lu, Quan
Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title_full Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title_fullStr Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title_full_unstemmed Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title_short Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts
title_sort rapid on-site detection of the bursaphelenchus xylophilus using recombinase polymerase amplification combined with lateral flow dipstick that eliminates interference from primer-dependent artifacts
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8971978/
https://www.ncbi.nlm.nih.gov/pubmed/35371187
http://dx.doi.org/10.3389/fpls.2022.856109
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