Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) as...
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KeAi Communications Co., Ltd.
2020-02-01
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| Series: | Journal of Integrative Agriculture |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2095311919626730 |
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| author | Yu LU Hai-jun ZHANG Zi-jing ZHAO Chang-long WEN Ping WU Shun-hua SONG Shuan-cang YU Lai-xin LUO Xiu-lan XU |
| author_facet | Yu LU Hai-jun ZHANG Zi-jing ZHAO Chang-long WEN Ping WU Shun-hua SONG Shuan-cang YU Lai-xin LUO Xiu-lan XU |
| author_sort | Yu LU |
| collection | DOAJ |
| description | Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) assay to droplet digital PCR (ddPCR) format for A. citrulli detection by optimizing reaction conditions. The performance of ddPCR in detecting A. citrulli pure culture, DNA, infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR, qPCR, and dilution plating method. The lowest concentrations detected (LCD) by ddPCR reached up to 2 fg DNA, and 102 CFU mL−1 bacterial cells, which were ten times more sensitive than those of the qPCR. When testing artificially infested watermelon and melon seed, 0.1% infestation level was detectable using ddPCR and dilution plating method. The 26 positive samples were identified in 201 commercial seed samples through ddPCR, which was the highest positive number among all the methods. High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future. |
| format | Article |
| id | doaj-art-fbf6dce6cf254fa78fe649671f2f96ab |
| institution | Kabale University |
| issn | 2095-3119 |
| language | English |
| publishDate | 2020-02-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj-art-fbf6dce6cf254fa78fe649671f2f96ab2025-08-20T03:58:04ZengKeAi Communications Co., Ltd.Journal of Integrative Agriculture2095-31192020-02-0119256156910.1016/S2095-3119(19)62673-0Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulliYu LU0Hai-jun ZHANG1Zi-jing ZHAO2Chang-long WEN3Ping WU4Shun-hua SONG5Shuan-cang YU6Lai-xin LUO7Xiu-lan XU8Beijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China; Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097, P.R.China; Correspondence LU Yu, Tel: +86-10-51503078Beijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China; Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097, P.R.China; Correspondence ZHANG Hai-jun, Tel: +86-10-51503078Beijing Key Laboratory of Seed Disease Testing and Control, College of Plant Pathology, China Agricultural University, Beijing 100093, P.R.ChinaBeijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.ChinaBeijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China; Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097, P.R.ChinaBeijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China; Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097, P.R.ChinaBeijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.ChinaBeijing Key Laboratory of Seed Disease Testing and Control, College of Plant Pathology, China Agricultural University, Beijing 100093, P.R.ChinaBeijing Key Laboratory of Vegetable Germplasms Improvement, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China; Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097, P.R.China; Correspondence XU Xiu-lan, Tel: +86-10-51503078Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seed-transmitted, so seed detection to prevent distribution of contaminated seed is crucial in disease management. In this study, we adapted a quantitative real-time PCR (qPCR) assay to droplet digital PCR (ddPCR) format for A. citrulli detection by optimizing reaction conditions. The performance of ddPCR in detecting A. citrulli pure culture, DNA, infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR, qPCR, and dilution plating method. The lowest concentrations detected (LCD) by ddPCR reached up to 2 fg DNA, and 102 CFU mL−1 bacterial cells, which were ten times more sensitive than those of the qPCR. When testing artificially infested watermelon and melon seed, 0.1% infestation level was detectable using ddPCR and dilution plating method. The 26 positive samples were identified in 201 commercial seed samples through ddPCR, which was the highest positive number among all the methods. High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.http://www.sciencedirect.com/science/article/pii/S2095311919626730bacterial fruit blotchAcidovorax citrullidroplet digital PCRseed detectionquantitative real-time PCR |
| spellingShingle | Yu LU Hai-jun ZHANG Zi-jing ZHAO Chang-long WEN Ping WU Shun-hua SONG Shuan-cang YU Lai-xin LUO Xiu-lan XU Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli Journal of Integrative Agriculture bacterial fruit blotch Acidovorax citrulli droplet digital PCR seed detection quantitative real-time PCR |
| title | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli |
| title_full | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli |
| title_fullStr | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli |
| title_full_unstemmed | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli |
| title_short | Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli |
| title_sort | application of droplet digital pcr in detection of seed transmitted pathogen acidovorax citrulli |
| topic | bacterial fruit blotch Acidovorax citrulli droplet digital PCR seed detection quantitative real-time PCR |
| url | http://www.sciencedirect.com/science/article/pii/S2095311919626730 |
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