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Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR

Vascular wilt, caused by Verticillium dahliae and V. longisporum, limits the quality and yield of agricultural crops. Although quantitative real-time PCR (qPCR) has greatly improved the diagnosis of these two pathogens over traditional, time-consuming isolation methods, the relatively poor detection...

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Autores principales: Wang, Di, Jiao, Xinya, Jia, Haijiang, Cheng, Shumei, Jin, Xi, Wang, Youhua, Gao, Yunhua, Su, Xiaofeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9441566/
https://www.ncbi.nlm.nih.gov/pubmed/36072220
http://dx.doi.org/10.3389/fcimb.2022.995705
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author Wang, Di
Jiao, Xinya
Jia, Haijiang
Cheng, Shumei
Jin, Xi
Wang, Youhua
Gao, Yunhua
Su, Xiaofeng
author_facet Wang, Di
Jiao, Xinya
Jia, Haijiang
Cheng, Shumei
Jin, Xi
Wang, Youhua
Gao, Yunhua
Su, Xiaofeng
author_sort Wang, Di
collection PubMed
description Vascular wilt, caused by Verticillium dahliae and V. longisporum, limits the quality and yield of agricultural crops. Although quantitative real-time PCR (qPCR) has greatly improved the diagnosis of these two pathogens over traditional, time-consuming isolation methods, the relatively poor detection sensitivity and high measurement bias for traceable matrix-rich samples need to be improved. Here, we thus developed a droplet digital PCR (ddPCR) assay for accurate, sensitive detection and quantification of V. dahliae and V. longisporum. We compared the analytical and diagnostic performance in detail of ddPCR and the corresponding qPCR assay against the genomic DNA (gDNA) of the two fungi from cultures and field samples. In our study, the species specificity, quantification linearity, analytical sensitivity, and measurement viability of the two methods were analyzed. The results indicated that ddPCR using field samples enhanced diagnostic sensitivity, decreased quantification bias, and indicated less susceptibility to inhibitors compared with qPCR. Although ddPCR was as sensitive as qPCR when using gDNA from cultures of V. dahliae and V. longisporum, its detection rates using field samples were much higher than those of qPCR, potentially due to the inhibition from residual matrix in the extracts. The results showed that digital PCR is more sensitive and accurate than qPCR for quantifying trace amounts of V. dahliae and V. longisporum and can facilitate management practices to limit or prevent their prevalence.
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spelling pubmed-94415662022-09-06 Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR Wang, Di Jiao, Xinya Jia, Haijiang Cheng, Shumei Jin, Xi Wang, Youhua Gao, Yunhua Su, Xiaofeng Front Cell Infect Microbiol Cellular and Infection Microbiology Vascular wilt, caused by Verticillium dahliae and V. longisporum, limits the quality and yield of agricultural crops. Although quantitative real-time PCR (qPCR) has greatly improved the diagnosis of these two pathogens over traditional, time-consuming isolation methods, the relatively poor detection sensitivity and high measurement bias for traceable matrix-rich samples need to be improved. Here, we thus developed a droplet digital PCR (ddPCR) assay for accurate, sensitive detection and quantification of V. dahliae and V. longisporum. We compared the analytical and diagnostic performance in detail of ddPCR and the corresponding qPCR assay against the genomic DNA (gDNA) of the two fungi from cultures and field samples. In our study, the species specificity, quantification linearity, analytical sensitivity, and measurement viability of the two methods were analyzed. The results indicated that ddPCR using field samples enhanced diagnostic sensitivity, decreased quantification bias, and indicated less susceptibility to inhibitors compared with qPCR. Although ddPCR was as sensitive as qPCR when using gDNA from cultures of V. dahliae and V. longisporum, its detection rates using field samples were much higher than those of qPCR, potentially due to the inhibition from residual matrix in the extracts. The results showed that digital PCR is more sensitive and accurate than qPCR for quantifying trace amounts of V. dahliae and V. longisporum and can facilitate management practices to limit or prevent their prevalence. Frontiers Media S.A. 2022-08-22 /pmc/articles/PMC9441566/ /pubmed/36072220 http://dx.doi.org/10.3389/fcimb.2022.995705 Text en Copyright © 2022 Wang, Jiao, Jia, Cheng, Jin, Wang, Gao and Su 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 Cellular and Infection Microbiology
Wang, Di
Jiao, Xinya
Jia, Haijiang
Cheng, Shumei
Jin, Xi
Wang, Youhua
Gao, Yunhua
Su, Xiaofeng
Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title_full Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title_fullStr Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title_full_unstemmed Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title_short Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR
title_sort detection and quantification of verticillium dahliae and v. longisporum by droplet digital pcr versus quantitative real-time pcr
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9441566/
https://www.ncbi.nlm.nih.gov/pubmed/36072220
http://dx.doi.org/10.3389/fcimb.2022.995705
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