Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides.
Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides.
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DOI：
10.1111/1574-6968.12453
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发表时间：
2014-06
影响因子：
2.1
通讯作者：
R. Takahashi;S. Fukuta;S. Kuroyanagi;N. Miyake;H. Nagai;K. Kageyama;Y. Ishiguro
中科院分区：
文献类型：
--
作者：
R. Takahashi;S. Fukuta;S. Kuroyanagi;N. Miyake;H. Nagai;K. Kageyama;Y. Ishiguro
一品红根腐病是由螺旋腐霉在高温下引起的，在世界许多地方都是一个严重的问题。我们已经开发了一种特异性的，环介导等温扩增（LAMP）检测这种病原体的快速诊断。使用核糖体DNA内转录间隔区序列设计引物。使用40种腐霉属物种，包括螺旋腐霉，11种疫霉属物种和8种其他土传病原体建立引物特异性。用螺旋藻基因组DNA进行了灵敏度试验，检出限为c。100 fg，这与聚合酶链反应（PCR）相当。此外，我们测试了病原体检测一品红根的难易程度。LAMP检测结果与常规平板法检测结果一致，且灵敏度高于PCR。因此，本研究建立的LAMP方法是一种快速、简便的螺旋体并殖吸虫检测方法。
Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides.