Development of a loop-mediated isothermal amplification method for the rapid detection of Pythium ultimum
Development of a loop-mediated isothermal amplification method for the rapid detection of Pythium ultimum
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开发用于快速检测终极腐霉的环介导等温扩增方法
DOI：
10.1007/s13313-017-0517-9
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发表时间：
2017-09
影响因子：
1.4
通讯作者：
Dou Daolong
中科院分区：
文献类型：
--
作者：
Shen Danyu;Li Qingling;Yu Jia;Zhao Yuanyuan;Zhu Ye;Xu Heng;Dou Daolong
腐霉是一种重要的土传病原菌，在许多植物中引起立枯病和根腐病。我们建立了一种环介导的等温扩增(LAMP)方法，用于快速、准确地检测最终P.ultimum。用比较基因组学方法从终极假单胞菌基因组中鉴定了一个编码孢子壁蛋白的目的基因。LAMP方法在℃下高效扩增60min，并对其特异性和敏感性进行了评价。在检测的病原菌中，只有P.ultimum分离物呈阳性结果。LAMP方法的检出限为1pgμL−1DNA，灵敏度为常规聚合酶链式反应的1000倍。此外，该方法还成功地检测了从受感染的植物组织中提取的P.ultimum DNA，植物中的P.ultimum的检出限与体外样品的检测限基本相同。因此，我们的LAMP检测方法能够快速、特异和灵敏地从纯培养物和受感染的植物组织中检测到最终疫霉的DNA。
Pythium ultimum is an important soil-borne pathogen that causes damping-off and root rot in a large number of plant species. We developed a loop-mediated isothermal amplification (LAMP) method for the rapid and accurate detection of P. ultimum. A target gene encoding a spore wall protein was identified from the P. ultimum genome using a comparative genomics approach. The LAMP assay efficiently amplified the target DNA at 64 °C for 60 min, and its specificity and sensitivity were evaluated. Of the pathogen isolates tested, positive results were obtained only with P. ultimum isolates. The detection limit of the LAMP method was 1 pg μL−1 DNA, which is 1000 times more sensitive than conventional PCR. Moreover, this method was successfully used to detect P. ultimum DNA extracted from infected plant tissues, and the detection limit for P. ultimum in plants was nearly the same as for in vitro samples. Consequently, our LAMP assay enables the rapid, specific, and sensitive detection of P. ultimum DNA from pure cultures, as well as from infected plant tissues.
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影响因子：
2.7
作者：
Buehlmann, A.;Pothier, J. F.;Duffy, B.
通讯作者：
Duffy, B.
影响因子：
2.1
作者：
Dai, Ting-Ting;Lu, Chen-Chen;Zheng, XiaoBo
通讯作者：
Zheng, XiaoBo
影响因子：
3.2
作者：
LARKIN, RP;ENGLISH, JT;MIHAIL, JD
通讯作者：
MIHAIL, JD
影响因子：
2.1
作者：
R. Takahashi;S. Fukuta;S. Kuroyanagi;N. Miyake;H. Nagai;K. Kageyama;Y. Ishiguro
通讯作者：
R. Takahashi;S. Fukuta;S. Kuroyanagi;N. Miyake;H. Nagai;K. Kageyama;Y. Ishiguro
影响因子：
2.7
作者：
T. Pettitt;A. Wakeham;M. Wainwright;J. G. White
通讯作者：
T. Pettitt;A. Wakeham;M. Wainwright;J. G. White