Visual detection of Didymella bryoniae in cucurbit seeds using a loop-mediated isothermal amplification assay

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• 作者：Yanli Tian ; Da Liu ; Yuqiang Zhao ; Jing Wu ; Baishi Hu…
• 关键词：Didymella bryoniae ; LAMP ; Visual detection
• 刊名：European Journal of Plant Pathology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：147
• 期：2
• 页码：255-263
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Plant Pathology; Plant Sciences; Ecology; Agriculture; Life Sciences, general;
• 出版者：Springer Netherlands
• ISSN：1573-8469
• 卷排序：147

文摘

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.