Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli  

作　　者：LU Yu ZHANG Hai-jun ZHAO Zi-jing WEN Chang-long WU Ping SONG Shun-hua YU Shuan-cang Luo Lai-xin XU Xiu-lan 

机构地区：[1]Beijing Key Laboratory of Vegetable Germplasms Improvement,Beijing Vegetable Research Center,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,P.R.China [2]Supervision,Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs,Beijing 100097,P.R.China [3]Beijing Key Laboratory of Seed Disease Testing and Control,College of Plant Pathology,China Agricultural University,Beijing 100093,P.R.China

出　　处：《Journal of Integrative Agriculture》2020年第2期561-569,共9页农业科学学报（英文版）

基　　金：supported by the the National Key Research and Development Program of China (2017YFD0201602);the National Natural Science Foundation of China (31401704);the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)

摘　　要：Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,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.Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide. The pathogen is seedtransmitted, 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.

关 键 词：bacterial fruit blotch Acidovorax citrulli droplet digital PCR seed detection quantitative real-time PCR 

分 类 号：S432.42[农业科学—植物病理学]