Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America.
United States Department of Agriculture, Agricultural Research Service, Harry K. Dupree-Stuttgart National Aquaculture Research Center, Stuttgart, Arkansas, United States of America.
PLoS One. 2021 Dec 13;16(12):e0250808. doi: 10.1371/journal.pone.0250808. eCollection 2021.
Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20-60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.
鲑鱼真菌病是淡水水产养殖中的一种重要疾病，与水霉属的卵菌病原体有关。早期检测到大量的水霉属病原体，将有助于做出明智的治疗决策，从而显著减少损失。本研究首次报道了用于检测水霉属的环介导等温扩增（LAMP）的开发，并将其与定量 PCR（qPCR）进行了比较。开发的方案针对核糖体 DNA 的内部转录间隔区（ITS）区域和细胞色素 C 氧化酶亚基 1（CoxI）基因，并且仅对水霉属表现出特异性。该 LAMP 方法可以检测低至 10 fg 的 S. salmonis DNA，而 qPCR 方法的检测限为 2 pg 的 S. salmonis DNA，表明 LAMP 比 qPCR 具有更高的灵敏度。当应用于检测水样中的病原体时，两种方法都可以在仅存在一个水霉属游动孢子时检测到病原体。我们提出 LAMP 作为一种快速（约 20-60 分钟）和敏感的分子诊断工具，用于检测水霉属，适用于现场应用。
