环介导等温扩增技术检测大丽轮枝菌

 本研究基于环介导等温扩增技术(loop-mediated isothermal amplification，LAMP)，通过比对分析大丽轮枝菌(Verticillium dahliae)与其相近种不同靶标序列间的差异，选取Gpd(glyceraldehyde-3-phosphate dehydrogenase，甘油醛-3-磷酸脱氢酶)基因作为靶标基因，设计并筛选了四条特异性强、灵敏度高的LAMP引物和两条环引物，建立了一种基于颜色判定的简单、快速和灵敏的大丽轮枝菌的检测方法，并进行了特异性、灵敏度实验及田间发病组织的检测。该方法在62 ℃等温条件下进行核酸扩增反应70 min，扩增前加入染料HNB(羟基萘酚蓝)，反应后根据染料颜色变化判定扩增结果。特异性试验中，仅含有大丽轮枝菌菌株DNA的反应管扩增后呈天蓝色的阳性反应，而其他供试菌株均呈紫色的阴性反应。该方法的最低检测限为100 pg·μL^-1，在土壤中检测的灵敏度为10个孢子/0.25g土壤。该技术能够检测出棉花发病组织中的目标菌，对采自江苏和山东的24份疑似病害样本进行检测，11份为阳性。该方法的建立为大丽轮枝菌的检测及其所致病害的诊断提供了新的技术。

Rapid detection of Verticillium dahliae using a loop-mediated isothermal amplification assay

Based on loop-mediated isothermal amplification technology, comparing Verticillium dahliae with approximate species among different target sequences, we selected the Gpd(glyceraldehyde-3-phosphate dehydrogenase) as the target gene, designed and selected four high specificity and sensitivity LAMP primers and two loop primers to develop a simple, rapid and sensitive method for the detection of V. dahliae. The LAMP assay efficiently amplified the target gene in 70 min at 62 ℃ and was evaluated for specificity, sensitivity and diseased cotton tissue. The result can be directly determined using naked eye by adding HNB (Hydroxynaphthol blue) before the amplification. The specificity was evaluated against V. albo-atrum, V. nigrescens and other fungi isolates. A positive color (sky blue) was only observed in the presence of V. dahliae, whereas all other isolates showed purple color as negative control. The detection limit of the LAMP assay for V. dahliae was 100 pg·μL^-1 and its sensitivity for detecting the pathogen in soil was 10 conidiospores in 0.25g soil. V. dahliae was detected in 11 diseased cotton from 24 suspect diseased samples collected from Jiangsu and Shandong provinces. This method provides a new technology for the detection of V. dahliae.