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Development of a new real-time TaqMan PCR assay for the detection of the Prunus pathogen Monilinia kusanoi
Australasian Plant Pathology ( IF 1.4 ) Pub Date : 2021-01-15 , DOI: 10.1007/s13313-021-00774-4
K. Dharmaraj , B. J. R. Alexander , M. Toome-Heller

Monilinia kusanoi causes shoot blight, leaf blight and fruit rot of several Prunus species, and is currently known to be present only in Japan and Korea. Despite the restricted distribution range, the risk remains that the pathogen could spread with the transportation of plant material, especially since no DNA based diagnostic methods are available to detect this fungus. In this study, a new TaqMan probe-based real-time assay was developed for specific and rapid detection of M. kusanoi. The assay targets the single copy elongation factor gene and enables the detection down to 1 pg of pathogen DNA in a plant sample, which corresponds to approximately 25 hyphal cells. When testing a panel of fungal cultures, all M. kusanoi isolates were detected and there was no cross-reaction with other Monilinia species or related fungi. The assay was duplexed with a plant internal control assay to allow simultaneous detection of the pathogen and cytochrome oxidase gene from host plants. This newly developed real-time PCR assay will facilitate rapid and sensitive pathogen screening of imported plant material, as M. kusanoi is a pathogen of biosecurity concern in New Zealand and Australia and would also be useful for research and screening purposes in areas where the pathogen is already established.



中文翻译:

开发一种新的实时TaqMan PCR检测试剂盒来检测李属病原菌

苦桑(Monilinia kusanoi)引起几种李属植物的枯萎病,叶枯病和果实腐烂,目前已知仅在日本和韩国存在。尽管分布范围受到限制,但病原体仍可能随植物材料的运输而传播,尤其是因为没有基于DNA的诊断方法可检测这种真菌。在这项研究中,开发了一种新的基于TaqMan探针的实时测定法,用于特异和快速检测kusanoi。该测定法以单拷贝延伸因子基因为目标,并能够检测植物样品中低至1 pg的病原体DNA,相当于大约25个菌丝细胞。当测试一组真菌培养物时,所有桑桑分枝杆菌分离物被检测到,与其他莫妮莉亚菌种或相关真菌没有交叉反应。该测定与植物内部对照测定双重进行,以允许同时检测来自宿主植物的病原体和细胞色素氧化酶基因。这种新开发的实时PCR分析法将有助于对进口植物材料进行快速,敏感的病原体筛查,因为kusanoi菌是新西兰和澳大利亚关注生物安全性的病原体,也可用于病原体地区的研究和筛查目的已经建立。

更新日期:2021-01-15
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