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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Frontiers Media S.A.
2022-08-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2022.995705/full |
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| author | Di Wang Xinya Jiao Haijiang Jia Shumei Cheng Xi Jin Youhua Wang Yunhua Gao Xiaofeng Su |
| author_facet | Di Wang Xinya Jiao Haijiang Jia Shumei Cheng Xi Jin Youhua Wang Yunhua Gao Xiaofeng Su |
| author_sort | Di Wang |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-13T18:41:48Z |
| format | Article |
| id | doaj.art-ac5f18b1596747e087635624c3db69fd |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-04-13T18:41:48Z |
| publishDate | 2022-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-ac5f18b1596747e087635624c3db69fd2022-12-22T02:34:42ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-08-011210.3389/fcimb.2022.995705995705Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCRDi Wang0Xinya Jiao1Haijiang Jia2Shumei Cheng3Xi Jin4Youhua Wang5Yunhua Gao6Xiaofeng Su7Center for Advanced Measurement Science, National Institute of Metrology, Beijing, ChinaCollege of Food Science and Technology, Hebei Agricultural University, Baoding, ChinaRaw Material Technology Center of Guangxi Tobacco, Nanning, ChinaCollege of Food Science and Technology, Hebei Agricultural University, Baoding, ChinaHebei Technology Innovation Center for Green Management of Soil-Borne Diseases, Baoding University, Hebei, ChinaBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, ChinaCenter for Advanced Measurement Science, National Institute of Metrology, Beijing, ChinaBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, ChinaVascular 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.https://www.frontiersin.org/articles/10.3389/fcimb.2022.995705/fulldroplet digital PCRquantitative real-time PCRsensitivityquantificationtolerance |
| spellingShingle | Di Wang Xinya Jiao Haijiang Jia Shumei Cheng Xi Jin Youhua Wang Yunhua Gao Xiaofeng Su Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR Frontiers in Cellular and Infection Microbiology droplet digital PCR quantitative real-time PCR sensitivity quantification tolerance |
| 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 | droplet digital PCR quantitative real-time PCR sensitivity quantification tolerance |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2022.995705/full |
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