Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.
Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.
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DOI：
10.1371/journal.pone.0111094
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Zhou MG
中科院分区：
文献类型：
--
作者：
Duan YB;Ge CY;Zhang XK;Wang JX;Zhou MG
灰葡萄孢 (Botrytis cinerea) 是一种毁灭性的植物病原体，会导致灰霉病。在本研究中，我们开发了一种基于 Bcos5 序列的灰霉病菌视觉检测方法，使用环介导等温扩增 (LAMP) 和羟基萘酚蓝染料 (HNB)。 LAMP 反应最佳温度为 63°C，反应时间为 45 分钟。当在扩增前添加 HNB 时，含有灰霉病菌 DNA 的样品在反应后呈现出特有的天蓝色，而不含 DNA 或含有其他植物病原真菌 DNA 的样品则不会。 LAMP产物进行凝胶电泳时，再次证实了HNB染色方法的结果。这种 LAMP 检测对灰霉病的检测限为每次反应 10−3 ng µL−1 基因组 DNA，比传统 PCR (10−2 ng µL−1) 的灵敏度高 10 倍。可以在接种的番茄和草莓花瓣中对灰霉病菌接种物进行 LAMP 检测。 191例病变标本中，LAMP检测阳性180例（94.2%），组织分离阳性172例（90.1%），PCR检测阳性147例（77.0%）。由于LAMP检测在灵敏度、特异性、重复性、可靠性和可视性方面表现良好，因此适用于在储存前和运输过程中快速检测受感染植物材料（例如切花、水果和蔬菜）中的灰霉病菌。
Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63°C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10−3 ng µL−1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10−2 ng µL−1). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.
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