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

Tian, Yanli, Liu, Da, Zhao, Yuqiang, Wu, Jing, Hu, Baishi, Walcott, R. R.
European journal of plant pathology 2017 v.147 no.2 pp. 255-263
Cucurbitaceae, DNA, DNA-directed RNA polymerase, Didymella bryoniae, detection limit, genes, loop-mediated isothermal amplification, muskmelons, pathogens, quantitative polymerase chain reaction, seeds, watermelons, China
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.